I n s t ru c t i o n s D AKO LSAB 2 System, P e r o x i d a s e

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1 I n s t ru c t i o n s D AKO LSAB 2 System, P e r o x i d a s e Universal Code No. K0672 K0673 K0675 Size 15 ml 15 ml 110 ml For Laboratory Use.

2 1. INTENDED USE FOR LABORATORY USE These instructions apply to the Universal DAKO Labelled Streptavidin-Biotin 2 System, Horseradish Peroxidase (DAKO LSAB 2 System, HRP). This system is intended for use with primary antibodies from RABBIT or MOUSE supplied by the user for the qualitative identification of antigens by light microscopy and immunohistochemistry in paraffin-embedded tissues, cryostat tissues and cell preparations to aid in the diagnoses of pathological disorders. Tissues processed in a variety of fixatives including ethanol, B-5, Bouin s, zinc formalin, and neutral buffered formalin may be used. 2. SUMMARY AND EXPLANATION Immunoenzymatic/immunohistochemical (IHC) staining techniques allow for the visualization of tissue (cell) antigens. These techniques are based on the immunoreactivity of antibodies and the chemical properties of enzymes or enzyme complexes which react with colorless substrate-chromogens to produce a colored end-product. Initial immunoenzymatic stains utilized the direct method which conjugated enzymes directly to an antibody with known antigenic specificity (primary antibody). Although this technique lacked the sensitivity of later methods, it allowed for the visualization of tissue antigens using a standard light microscope. The sensitivity of IHC stains was significantly improved with the development of an indirect technique. In this two-step method, enzyme-labeled secondary antibodies react with the antigen-bound primary antibody. A further increase in sensitivity over the indirect technique was achieved with the introduction of the peroxidase-antiperoxidase (PAP) enzyme c o m p l e x. 1 In this method the secondary antibody serves as a linking antibody between the primary antibody and the PAP c o m p l e x. 1 Subsequent developments in IHC exploited the strong affinity of avidin for biotin and resulted in the avidinbiotin complex (ABC) method of Hsu et al. 2 This technique employs an enzyme labeled avidin-biotin complex which is mixed prior to use and forms a complex with a biotinylated secondary antibody. The ABC method increased reagent sensitivity when compared to the PAP method. The DAKO LSAB 2 System, HRP is based on a modified labeled avidin-biotin (LAB) technique in which a biotinylated secondary antibody forms a complex with peroxidaseconjugated streptavidin molecules. 3 In comparison to the ABC method, the LAB/LSAB method has been reported to be four to eight times more sensitive. 4 Giorno, et al 4, attribute the increased sensitivity to the smaller size of the enzyme-labelled (strept)avidin complex of the LAB/LSAB method when compared to the avidin-biotin enzyme complex of the ABC method. Endogenous avidin binding activity (EABA) has been noted in frozen sections of liver (entire hepatic nodule) and kidney (tubular epithelium), as well as in frozen and formalin-fixed lymphoid tissues (paracortical histiocytes). 5-8 EABA can be suppressed by sequential 20 minute incubations, first with 0.1% avidin and then with 0.01% biotin in 0.05M Tris HCl buffer, ph , prior to Section 9.B. Step 1, or with DAKO Biotin Blocking System (Code No. X0590). Endogenous peroxidase or pseudoperoxidase activity can be found in hemoproteins such as hemoglobin, myoglobin, cytochrome and catalase as well as in eosinophils. 6, 9 In formalin fixed tissue this activity can be inhibited by incubating specimens with 3% hydrogen peroxide for five minutes prior to application of the primary antibody. Blood and bone marrow smears and frozen tissues can be treated with DAKO Peroxide Blocking Reagent (Code No. S2001). However, this procedure does not abolish the reddish-brown pigment of hemoproteins. Alternately, a solution of methanol-hydrogen peroxide can be used. Some antigens may become denatured with this procedure.

3 The clinical interpretation of any positive staining or its absence should be complemented by morphological and histological studies with proper controls. Evaluations should be made within the context of the patient s clinical history and other diagnostic tests by a qualified individual. Tissues from persons infected with hepatitis B virus and containing hepatitis B surface antigen (HBsAg) may exhibit nonspecific staining with horseradish peroxidase. 3. PRINCIPLES OF PROCEDURE The DAKO LSAB 2 System, HRP is a sensitive and versatile IHC procedure which permits the simultaneous processing of numerous specimens with rabbit or mouse primary antibodies in less than one hour. Endogenous peroxidase activity is quenched by incubating the specimen for 5 minutes with 3% hydrogen peroxide. The specimen is then incubated with an appropriately characterized and diluted rabbit or mouse primary antibody, followed by sequential 10-minute incubations with a biotinylated link antibody (containing anti-rabbit and anti-mouse immunoglobulins) and peroxidase-labelled streptavidin. Staining is completed after an incubation with the Substrate Chromogen (AEC or DAB). For AEC in K0672, this is a 10-minute incubation with 3-amino-9-ethylcarbazole (AEC) Substrate-Chromogen which results in a red-colored precipitate at the antigen site. For DAB in K0673, this is a 5 10-minute incubation with 3-3' diaminobenzidine (DAB) Substrate-Chromogen which results in a brown-colored precipitate at the antigen site. 4. MATERIALS A. Reagents Provided Code No. K0672: The following materials, sufficient for 150 tissue sections, based upon 100 µl per section, are included in this kit: Bottle No. Quantity Description 1 1x15 ml Hydrogen Peroxide: 3% hydrogen peroxide in water. 2 1x15 ml Link Antibody: biotin labelled affinity isolated goat anti-rabbit and goat anti-mouse immunoglobulins in phosphate buffered saline (PBS), containing carrier protein and M sodium azide. 3 1x15 ml Streptavidin-HRP: Streptavidin conjugated to horseradish peroxidase in PBS containing carrier protein and anti-microbial agents. 4 1x15 ml AEC Substrate Chromogen: AEC in N,Ndimethylformamide (DMF) and acetate buffer, ph 5.0, containing hydrogen peroxide, stabilizers, enhancers and an anti-microbial agent. Keep at 2 8 C. Code No. K0673: The following materials, sufficient for 150 tissue sections, based upon 100 µl per section, are included in this kit: Bottle No. Quantity Description 1 1x15 ml Hydrogen Peroxide: 3% hydrogen peroxide in water. 2 1x15 ml Biotinylated Link: biotin labelled affinity isolated goat anti-rabbit and goat anti-mouse immunoglobulins in phosphate buffered saline (PBS), containing carrier protein and M sodium azide. 3 1x15 ml Streptavidin-HRP: Streptavidin conjugated to horseradish peroxidase in PBS containing carrier protein and anti-microbial agents. 4 a 1x15 ml Buffered Substrate: Imidazole-HCl buffer ph 7.5 containing hydrogen peroxide and an anti-microbial agent. 4 b 1x1 ml DAB Chromogen: 3,3'-diaminobenzidine in chromogen solution.

4 ACCESSORIES 1 Calibrated test tube 1 Plastic Pasteur pipette Code No. K0675(11): The following materials, sufficient for 1100 tissue sections, based upon 100 µl per section, are included in this kit: Bottle No. Quantity Description 1 1x110 ml Biotinylated Link: biotin labelled affinity isolated goat anti-rabbit and goat anti-mouse immunoglobulins in phosphate buffered saline (PBS), containing carrier protein and M sodium azide. 2 1x110 ml Streptavidin-HRP: Streptavidin conjugated to horseradish peroxidase in PBS containing carrier protein and anti-microbial agents. Code No. K0675(89): The following materials, packaged for use with the DAKO Autostainer (Code No. S3400), are included in this kit: Quantity 10x11 ml 10x11 ml Description Biotinylated Link: biotin labelled affinity isolated goat anti-rabbit and goat anti-mouse immunoglobulins in phosphate buffered saline (PBS), containing carrier protein and M sodium azide. Streptavidin-HRP: Streptavidin conjugated to horseradish peroxidase in PBS containing carrier protein and anti-microbial agents. *DAKO Autostainer is available in North and South Americas, Australia and New Zealand only. B. Materials Required but Not Supplied Absorbent wipes Ammonium hydroxide, 15 M diluted to 37 mm Antibodies: DAKO N-Series ready-to-use or concentrated antibodies diluted in DAKO antibody diluent, Code Nos. S0809 or S3022 (see Section 6.B.) Antibody diluent, DAKO Code No. S0809 or S3022 (see Section 6.B.) Control tissues, positive and negative Counterstain; aqueous based, such as Mayer s hematoxylin (Lillie s modification), DAKO Code No. S3309 (see Section 6.E.) Coverslips Distilled water Drying oven, capable of maintaining 60 C or less. Ethanol, absolute and 95% Humid Chamber (optional) Light microscope (20x 800x) Mounting media, such as DAKO Faramount (Code No. S3025) or DAKO Glycergel (Code No. C0563) Negative control reagents for concentrated primary antibodies (see Sections 6.C. and 10.C.) Slides, Poly L-lysine coated or DAKO Silanized (Code No. S3003) (see Section 8.A., Adherence of Paraffin-Embedded Tissue Sections to Microscope Slides) Staining jars or baths Timer (capable of 2 10 minute intervals) Wash bottles

5 Wash buffer solution (see Section 6.A.) Xylene, toluene or xylene substitutes For the K0675 LSAB 2 System (110 ml), the following reagents are required in addition to the above list: Hydrogen peroxide, 3% solution Substrate-Chromogen solution such as DAKO AEC Substrate-Chromogen (Code No. K3464, 110 ml, ready-to-use) or DAKO Liquid DAB (Code No. K3466) C. Optional Materials, Not Supplied Buffers: Phosphate buffered saline (PBS, DAKO Code No. S3024) Tris-buffered saline (TBS, DAKO Code No. S3001, or S1968) TBST (Tris-buffered saline with Tween 20, DAKO Code No. S3306) Proteolytic enzymes: Pepsin (DAKO Code No. S3002) Proteinase K (DAKO Code No. S3004 or S3020) Trypsin (DAKO Code No. S2012) Others: Positive Control Slides (DAKO Code No. T1064 or T1065) Target retrieval solution (DAKO Code no. S1699 or S1700) Target retrieval solution, High ph (DAKO Code No. S3307 or S3308) 5. PRECAUTIONS A. Product Specific 1. FOR LABORATORY USE. 2. This product contains sodium azide (NaN 3 ), a chemical highly toxic in pure form. At product concentrations, though not classified as hazardous, build-ups of sodium azide may react with lead and copper plumbing to form highly explosive metal oxides. Upon disposal, flush with large volumes of water to prevent azide build-up in plumbing. 10,11 3. K0672 contains 3-amino-9-ethylcarbazole (AEC) and N,Ndimethylformamide (DMF). At product concentrations, AEC is not classified as a hazardous chemical. DMF, used as a solvent for AEC, is classified as a teratogen. Pregnant workers should avoid exposure to the AEC/DMF solution at all times. The AEC/DMF solution may cause irritation upon skin contact. If skin contact occurs, immediately flush affected area with soap and water. 4. K0673 contains 3,3'-diaminobenzidine (DAB) and may be harmful by inhalation, in contact with skin and if swallowed. Material is irritating to eyes and skin. If skin contact should occur, flush affected area with soap and water. Note: Although Diaminobenzidine is structurally related to Benzidine, there is no evidence for the carcinogenicity of Diaminobenzidine. 5. The AEC and DAB Substrate-Chromogens are sensitive to contamination from a variety of oxidizing agents such as metals, bacteria, dust and commonly used laboratory glassware. To avoid contamination and premature expiration, avoid exposing the AEC or DAB solutions to any potential source of contamination and never pipette directly from the bottle. Pour out required amount into a clean container and pipette from it. Do not return excess AEC or DAB solution to the primary storage container. 6. The reagents provided are optimally diluted. Further dilution may result in loss of antigen staining. Any such change must be validated by the user. Differences in tissue processing and technical procedures in the user s laboratory may produce significant variability in results necessitating regular performance of in-house controls (see Section 10, Quality Control).

6 7. Do not substitute reagents from other lot numbers or from kits of other manufacturers. 8. Incubation times or temperatures other than those specified may give erroneous results; any such changes must be validated by the user. 9. Enzymes and chromogens may be affected adversely if exposed to excessive light levels. Do not store kit components or perform staining in strong light, such as direct sunlight. 10.Wear appropriate Personal Protective Equipment to avoid contact with eyes and skin. Unused reagents should be disposed of according to local, state, and federal regulations. Refer to the Material Safety Data Sheet (MSDS) for additional information. B. General 1. Specimens, before and after fixation, and all materials exposed to them, should be handled as if capable of transmitting infection and disposed of with proper precautions. 12 Never pipette reagents by mouth and avoid contacting the skin and mucous membranes with reagents and specimens. If reagents or specimens come in contact with sensitive areas, wash with copious amount of water. 2. Inhalation or ingestion of xylene or the highly allergenic fixative formaldehyde is harmful. Prepare in fume hood. If swallowed, induce vomiting. If skin or eye contact occurs, wash thoroughly with water. 3. Organic reagents are flammable. Do not use near open flame. 4. Minimize microbial contamination of reagents or incorrect results may occur. 5. Avoid splashing of reagents or generation of aerosols. 6. Avoid physical contact and inhalation of 15M ammonium hydroxide. Prepare in fume hood. If skin or eye contact occurs, wash thoroughly with water. 7. When using mercuric-chloride fixatives such as B-5 or Zenkers, additional safety precautions are necessary. 6. REAGENT PREPARATION It is convenient to prepare the following reagents prior to staining. A. Wash Buffer Solution 0.02 M Phosphate Buffered Saline (DAKO PBS, Code No. S3024), 0.05 M Tris-HCl buffer or 0.05 M Tris Buffered Saline ( D A K O TBS, Code No s. S3001, or S1968), ph , without sodium azide, are suitable wash buffer solutions. Wash buffer solutions containing sodium azide inactivate horseradish peroxidase and result in negative staining. To reduce excessive nonspecific staining, DAKO TBST (Tris buffered saline with Tween 20, Code No. 3306) or 0.05M Tris containing 0.3M NaCl and 0.1% Tween 20 (DAKO Code No. S1966), ph 7.6 is recommended for use as a wash buffer. Store unused buffer at 2 8 C. Discard buffer if cloudy in a p p e a r a n c e. Distilled water may be used for rinsing the hydrogen peroxide, substrate-chromogen solution, and counterstain. B. Primary Antibody A wide range of DAKO N-series ready-to-use P r i m a r y Antibodies and Negative Control Reagents are available for use with DAKO LSAB 2 Systems. Concentrated antibodies are also available from DAKO; however, optimal dilutions must be determined experimentally by the user. Dilutions should be prepared using DAKO Antibody Diluent, (Code No. S0809) which contains 0.05M Tris-HCl buffer, ph , and 1% bovine serum albumin (BSA). The BSA acts as a protein block and eliminates the need for a separate incubation with protein block reagent. DAKO Antibody Diluent with background reducing Components (Code No. S3022) is also suitable for use as a diluent. Diluent without protein carrier is not recommended. For most primary antibodies used with the LSAB2 Systems, an incubation of 10 minutes is sufficient.

7 C. Negative Control Reagent Ideally, a negative control reagent contains antibody which exhibits no specific reactivity with human tissues (non-human reactive) in the same matrix/solution as the primary antibody. The non-human reactive antibody should be the same subclass and animal species as the primary antibody, diluted to the same immunoglobulin or protein concentration as the diluted primary antibody using the same diluent. Depending on the type of primary antibody/antiserum used (see table in Section 10.C.), normal/non-immune serum from the same species as the primary at a protein concentration equivalent to the diluted primary in the same diluent may be suitable for use. The incubation period for the negative control reagent should correspond to that of the primary antibody/antiserum. When using DAKO N-Series ready-to-use primary antibodies optimized for use in DAKO LSAB 2 Systems, use the provided negative control reagent. For specific information regarding negative control reagent preparation, refer to Quality Control, Section 10.C. D. Substrate-Chromogen Solution The K0672 LSAB 2 System contains ready-to-use AEC, which is ready to apply to tissue or cell specimens. The K0673 L S A B 2 System contains DAB which is prepared as follows: Add 1 drop (or 20 µl) of the DAB Chromogen per ml of Buffered Substrate. Use the provided graduated test tube to measure the amount of Buffered Substrate needed. Mix well and apply solution using the provided transfer pipette. After use, rinse graduated test tube and pipette thoroughly with distilled water. Unused working DAB solution is stable for up to 2 weeks if stored at 2 8 C. If precipitate forms, mix well before using. D A K O Ready-to-use AEC Substrate-Chromogen Solution (Code No. K3464, 110 ml) or DAKO Liquid DAB (Code No. K3466) is recommended for use with the DAKO LSAB 2 System, HRP, 110 ml (Code No. K0675). Please follow the instructions provided with each substrate-chromogen system for substrate-chromogen preparation. E. Counterstain DAB chromogen yields an alcohol insoluble end-product and can be used with an alcohol-based hematoxylin. When AEC Substrate-chromogen is used, the colored end-product of the staining reaction is alcohol soluble and should only be used with aqueous-based counterstains such as Mayer s hematoxylin. Follow counterstaining of hematoxylin with a thorough rinse in distilled water, then immerse tissue slides into a bath of 37mM ammonia water (see Section 9.B. Step 6). Thirty-seven millimolar (37mM) ammonia water is prepared by mixing 2.5 ml of 15M (concentrated) ammonium hydroxide with 1 liter of water. Unused 37mM ammonia water may be stored at room temperature (20 25 C) in a tightly capped bottle for up to 12 months. Consult manufacturers guidelines for alternative counterstaining procedures. F. Mounting Media DAKO Faramount Aqueous Mounting Medium, ready-to-use (Code No. S3025) or DAKO Glycergel Mounting Medium (Code No. C0563) is recommended for aqueous mounting. Liquefy DAKO Glycergel by warming to approximately 40 ±5 C prior to use. 7. STORAGE AND HANDLING Reagents of the DAKO LSAB 2 System, HRP are to be stored at 2 8 C. Do Not Freeze. DAKO LSAB 2 Systems are suitable for use twelve (12) months from the date of manufacture when stored at 2 8 C. Do not use after the expiration date printed on reagent vials

8 and kit label. If reagents are stored under any conditions other than those specified, they must be validated by the user. 8 The AEC and DAB Substrate-Chromogen solutions are unstable at temperatures greater than 8 C. Use and store the AEC and DAB Substrate-Chromogen solutions at the recommended 2 8 C temperature range. These solutions may be used immediately after removal from the refrigerator. After use, return to 2 8 C storage as soon as possible. There are no obvious signs to indicate instability of these products. Therefore, positive and negative controls should be tested simultaneously with patient specimens. If unexpected staining is observed which cannot be explained by variation in laboratory procedures and a problem with the kit is suspected, contact DAKO Technical Services at 800/ SPECIMEN PREPARATION Prior to IHC staining, tissues must be fixed and processed. Fixation prevents autolysis and putrefaction of excised tissues, preserves antigenicity, enhances the refractive index of tissue constituents and increases the resistance of cellular elements to tissue processing. Tissue processing includes dehydration, clearing of dehydrating agents, infiltration of embedding media, embedding and sectioning of tissues. The most common fixatives for IHC tissue preparations are discussed below. These are guidelines only. Optimal procedures must be determined and verified by the user. Consult references 5, 6, 9, for specific information regarding tissue fixation and processing. A. Paraffin-Embedded Tissue General Comments Although 10% neutral phosphate-buffered formalin is the most common fixative, the DAKO LSAB 2 System, HRP has been successfully used with tissues processed in a variety of fixatives. Consequently, the choice of fixative is dependent on the limitations of the primary antibody and the user s institutional or laboratory constraints. Survival of tissue antigens for immunological staining may depend on the type and concentration of fixative, on fixation time, and on the size of the tissue specimen to be fixed. 1 5, 1 6 It is important to maintain optimal, standardized fixation conditions whenever possible in order to obtain reproducible staining. Where possible, use of thinner specimens coupled with shorter fixation times is recommended. Prolonged exposure to fixatives may result in the masking of antigens and contribute to reduced staining. Bouin s, B-5 and Zenker s fluid are alternative fixatives for the preservation of tissue antigens sensitive to routine formalin fixation (10% neutral buffered formalin). 1 6 Consult references 5, 6, 9, 14 16, the primary antibody specification sheet(s) and the protocol(s) supplied with the fixing reagent(s) for additional information regarding tissue fixation. Tissue Fixation in Formaldehyde-Based Solutions (Neutral Buffered Formalin, Zinc Formalin & Bouin s) Most formaldehyde-based fixatives contain 10% formalin, a neutral salt to maintain tonicity and a buffering system to maintain ph. These fixatives are well tolerated by tissues, exhibit good histological penetration and are well suited for labelled streptavidin-biotin immunostains. However, shrinkage or distortions may occur in poorly fixed and embedded tissue specimens. Fix small blocks of tissue (10 x 10 x 3mm) in 5 10 ml of neutral buffered formalin per block for up to 24 hours. Bouin s solution is an alternative formaldehyde based fixative which contains picric acid and is suitable for use on all tissues except kidney. Specimens may be fixed from 1 12 hours depending on tissue thickness. Excessively fixed tissues become brittle and adversely affect the appearance and quantity of lipids. Complete fixation with a 70% ethanol wash to precipitate soluble picrates prior to aqueous washes. Caution: dry picric acid crystals are highly explosive.

9 Follow fixation with dehydration, clearing, infiltration, and embedding. (See Section 8.A., Processing and Paraffin- Embedding) Mercuric-Chloride Containing Fixatives (B-5 and Zenker s) Mercuric-chloride fixatives, such as B-5 and Zenker s, frequently include a neutral salt to maintain tonicity and may be mixed with other fixatives. In general, mercuric-chloride fixatives are poor histological penetrators and are not well tolerated by tissue specimens. Consequently, small tissue blocks (7 x 7 x 2.5mm) and short fixation periods (1 6 hours for B-5, and 2 15 hours for Zenker s) are recommended. After fixation, the tissue block(s) should be rinsed well with water and placed in 70% ethanol for wet storage or until tissue processing can be completed. Conclude fixation with tissue processing and paraffin embedding. (See Section 8.A., Processing and Paraffin-Embedding) Prior to immunostaining, clear tissue sections of mercury deposits using an iodine/ethanol/sodium thiosulfate solution. 6,14 Tissue Fixation in Ethanol Ethanol is not widely employed as a fixative for routine histological techniques due to its poor penetrating ability. However, small pieces of tissue are fixed rapidly and show good cytological preservation. Fix tissue blocks (5 x 5 x 2mm) in absolute alcohol for 48 hours at room temperature (20 25 C), followed by two 1-hour baths in fresh xylene and two 1-hour baths in liquid paraffin. Follow paraffin infiltration with embedding. Processing and Paraffin Embedding After fixation, processing may be completed using an automatic tissue processor. Tissues are dehydrated using graded alcohols, cleared with xylene or xylene substitute, and infiltrated with paraffin wax. The tissue is subsequently embedded with paraffin wax in molds or cassettes which facilitate tissue sectioning. To minimize denaturing of antigens, do not expose tissues to temperatures in excess of 60 C during processing. Tissue blocks may be stored or sectioned on completion of embedding. Properly fixed and Paraffin-Embedded tissues will keep indefinitely if stored at 2 25 C. Adherence of Paraffin-Embedded Tissue Sections to Microscope Slides Collect sectioned tissues from Paraffin-Embedded blocks on clean glass slides. Dehydrate in an oven for one to two hours at 60 C or less. For increased adhesion of tissue sections during IHC staining procedures, use of poly-l-lysine coated slides or D A K O Silanized Glass Slides (Code No. S3003) is suggested. Coated slides are strongly recommended for staining procedures requiring proteolytic digestion or target retrieval. Slides with Paraffin-Embedded tissue sections can be kept indefinitely if stored at 2 25 C. Deparaffinization and Rehydration Prior to staining, tissue slides must be deparaffinized to remove embedding media and then rehydrated. Avoid incomplete removal of paraffin. Residual embedding media will result in increased nonspecific staining. 1. Place slides in a xylene bath and incubate for 5 (±1) minutes. Change baths and repeat once. 2. Tap off excess liquid and place slides in absolute ethanol for 3 (±1) minutes. Change baths and repeat once. 3. Tap off excess liquid and place slides in 95% ethanol for 3 (±1) minutes. Change baths and repeat once. 4. Tap off excess liquid and place slides in distilled or deionized water for a minimum of 30 seconds. Unless proteolytic digestion or target retrieval is required, commence staining procedure as outlined in Section 9.B.

10 Xylene and alcohol solutions should be changed after 40 slides. Toluene or xylene substitutes such as Histoclear may be used in place of xylene. If necessary, rehydrated tissues may be kept in buffer solution at 2 8 C for up to 18 hours prior to use. Allow tissues to come to room temperature (20 25 C) before staining. Proteolytic Digestion and Target Retrieval Formaldehyde is known to induce conformational changes in the antigen molecules by forming intermolecular crosslinkages. Excessive formalin fixation can mask antigenic sites and diminish specific staining. However, these sites may be revealed with proteolytic digestion or target retrieval of tissue sections prior to immunostaining. To determine if pretreatment of tissues is warranted, consult the specification sheet provided with each primary antibody. Deparaffinized and rehydrated tissue sections can be digested for 6 minutes at room temperature (20 25 C) in a solution containing 0.025% Protease Type XXIV (Sigma Code No. P-8038) and 0.025% CaCl 2 in Tris-HCl buffer, ph Rinse thoroughly with distilled water and continue with the staining procedure as outlined in Section 9.B. Other proteolytic enzymes, such as pepsin (DAKO Code No. S3002), pronase, proteinase K (DAKO Code No. S3004 or S3020), or trypsin (DAKO Code No. S2012) can also be used. Note: Overdigestion may result in nonspecific staining and/or unacceptable morphology. Target retrieval prior to immunostaining increases the staining intensity of many primary antibodies. For some antibodies, this procedure may be necessary. The retrieval procedure involves immersion of tissue sections into a target retrieval solution (DAKO Target Retrieval Solution, Code No. S1700 or DAKO Target Retrieval Solution, 10x concentrate, Code No. S1699) and heating. Rinse thoroughly with 0.05M Tris- HCl and continue with the staining procedure outlined in Section 9.B. Note: Fatty tissues may detach from poly-l-lysine coated slides during target retrieval. Silanized or charged slides are preferable for use. B. Frozen Tissue Frozen sections should be cut from snap-frozen tissue blocks (approximately 1.0 x 1.0 x 0.5cm) and air dried for 2 24 hours. Dried sections can be fixed in room temperature (20 25 C) acetone for 10 minutes or in buffered formolacetone for 30 seconds. Allow sections to air-dry until completely dehydrated. Proceed with immunostaining or wrap slides in aluminum foil and store at -20 C or lower for up to three to six months. Equilibrate wrapped, frozen sections to room temperature prior to use. If sections are too thick (greater than 4µm), incorrectly fixed, or unevenly dried, artifacts may result and interfere with interpretation of staining. This includes rupturing of cell membranes and chromatolysis. Nuclei may appear swollen and uniformly blue when counterstained with hematoxylin. Frozen tissues may be damaged by the hydrogen peroxide applied in Step 1 of the Staining Protocol (Section 9.B.). The purpose of this step is to quench nonspecific reactivity due to endogenous peroxidase enzymes. When using frozen tissues, omit the quench step. If necessary, substitute an alternative blocking reagent, DAKO Peroxidase Blocking Reagent (Code No. S2001), to minimize nonspecific staining. C. Other Specimens These systems may also be used for staining antigens in bone sections, bone marrow, blood smears, cytospins and imprints. Smears may be air-dried for 2 24 hours and processed for immediate staining or wrapped in aluminum foil and stored at -20 C or lower for up to three to six months. Air-dried or thawed smears may be fixed for 90 seconds in acetonemethanol (1:1). Fixation in acetone or acetone-methanolformalin (10:10:1) is also acceptable.

11 Osseous tissues must be decalcified prior to sectioning and processing to facilitate tissue cutting and prevent damage to 5, 9,17 microtome blades. Cell smears of peripheral blood, bone marrow and other body fluids containing a large number of hematopoietic cells may be damaged by the hydrogen peroxide applied in Step 1 of the Staining Protocol (Section 9.B.). The purpose of this step is to quench nonspecific reactivity due to endogenous peroxidase enzymes. When using cell preparations, omit the quench step. If necessary, substitute an alternative blocking reagent, DAKO Peroxidase Blocking Reagent (Code No. S2001), to minimize nonspecific staining. 9. STAINING PROCEDURE A. Procedural Notes The user should read these instructions carefully and become familiar with the system contents prior to use. Condensed instructions appear on the inside cover of the system box and are keyed to the colors and numbers of each reagent. See Section 5 for precautions. The reagents and instructions supplied in this system have been designed for optimal performance. Further dilution of the system reagents or alteration of incubation times or temperatures may give erroneous results. All system reagents except AEC Substrate-Chromogen should be equilibrated to room temperature (20 25 C) prior to immunostaining. Likewise, all incubations should be performed at room temperature. The AEC Substrate-Chromogen may be used immediately after removal from cold storage and does not have to be at room temperature prior to use. Return to 2 8 C for storage after use. Storage at temperatures above 8 C adversely affects the stability of the AEC Substrate-Chromogen. Do not allow tissue sections to dry during the staining procedure. Dried tissue sections may display increased nonspecific staining. Cover slides exposed to drafts. If prolonged incubations are used, place tissues in a humid environment. If the staining protocol must be interrupted, slides may be kept in a buffer bath following incubation of the link antibody (Step 3) for up to one hour at room temperature (20 25 C) without affecting staining performance. The sensitivity of the DAKO LSAB 2 System, HRP can be further increased by lengthening the incubation times of Steps 2, 3, and 4 to 30 ±5 minutes each. B. Staining Protocol STEP 1 HYDROGEN PEROXIDE Tap off excess liquid. Using a lintless tissue (such as a Kimwipe or gauze pad), carefully wipe around the specimen to remove any remaining liquid and to keep reagents within the prescribed area. Apply enough Hydrogen Peroxide to cover specimen. Incubate 5 (±1) minutes. Rinse gently with distilled water or buffer solution from a wash bottle (do not focus flow directly on tissue) and place in fresh buffer bath. STEP 2 PRIMARY ANTIBODY OR NEGATIVE CONTROL REAGENT Tap off excess liquid and wipe slides as before. Apply enough Primary Antibody or Negative Control reagent to cover specimen. Incubate 10 (±1) minutes unless otherwise specified. Rinse gently with buffer solution from a wash bottle (do not focus flow directly on tissue) and place in fresh buffer bath. STEP 3 BIOTINYLATED LINK Immediately tap off excess buffer and wipe slides as before. Apply enough YELLOW drops from Link Antibody to cover specimen.

12 Incubate 10 (±1) minutes. Rinse slides as in Step 2. If the staining protocol must be interrupted, slides may be kept in a buffer bath following incubation of the link antibody (Step 3) for up to one hour at room temperature (20 25 C) without affecting staining performance. STEP 4 STREPTAVIDIN-HRP Wipe slides as before. Apply enough RED drops of the Streptavidin reagent to cover specimen. Incubate 10 (±1) minutes. Rinse slide as before. STEP 5 SUBSTRATE-CHROMOGEN SOLUTION Remove AEC or DAB Substrate-Chromogen solutions from 2 8 C storage. For DAB preparation, refer to Section 6.D. Wipe slide as before. Apply enough of the AEC or DAB Substrate- Chromogen solution to cover specimen. Return the AEC or DAB Substrate-Chromogen to refrigerated storage. Incubate for 10 (±1) minutes for AEC and 5 10 minutes for DAB. Rinse gently with distilled water from a wash bottle (do not focus flow directly on tissue). Collect AEC or DAB Substrate-Chromogen waste in a hazardous materials container for proper disposal. STEP 6 HEMATOXYLIN COUNTERSTAIN (OPTIONAL) Immerse slides in a bath of hematoxylin. Incubate for two to five (2 5) minutes, depending on the strength of hematoxylin used. Rinse gently in a distilled water bath. Dip slides 10 times into a bath of 37mM ammonia water. (See Section 6.E.) Rinse slides in a bath of distilled or deionized water for two to five (2 5) minutes. STEP 7 MOUNTING Specimens may be mounted and coverslipped with an aqueous-based mounting medium such as DAKO Faramount (Code No. S3025) or DAKO Glycergel (Code No. C0563). Note: The AEC reaction product is soluble in organic solvents and is therefore not compatible with toluene- or xylene-based, permanent mounting media. Note: DAB may be mounted with any permanent mounting media. Note: Slides may be read when convenient. However, some fading may occur if slides are exposed to strong light over a period of one week. To minimize fading, store slides in the dark at room temperature (20 25 C). 10. QUALITY CONTROL Differences in tissue processing and technical procedures in the user s laboratory may produce significant variability in results, necessitating regular performance of in-house controls in addition to the following procedures. Consult the quality control guidelines of the College of American Pathologists (CAP) Certification Program for Immunohistochemistry and references for additional information. Refer to the package insert of each primary antibody used for details regarding sensitivity and immunoreactivity. A. Positive Control Tissue Controls should be fresh surgical specimens or autopsy tissues fixed, processed and embedded as soon as possible in the same manner as the patient sample(s). Positive control

13 tissues are indicative of correctly prepared tissues and proper staining techniques. One positive control tissue for each set of test conditions should be included in each staining run. Positive control tissues should also be weakly positive for each primary antibody to detect changes in reagent sensitivity. Commercially available tissue slides (such as DAKO C o n t r o l Slides, Code Nos. T1064 and T1065), or specimens processed differently from the patient sample(s) validate reagent performance only, and do not verify tissue preparation. Known positive control tissues should only be utilized for monitoring the correct performance of processed tissues and test reagents, NOT as an aid in formulating a specific diagnosis of patient samples. If the positive control tissues fail to demonstrate positive staining, results with the test specimens should be considered invalid. B. Negative Control Tissue One negative control tissue fixed, processed and embedded in a manner identical to the patient sample(s) should be stained with each primary antibody used in each staining run to verify the specificity of each primary antibody. This tissue should not exhibit specific staining and serves as an indicator of nonspecific staining. Negative control tissues should be used as an interpretive aid to distinguish specific staining from non-specific staining results. The variety of different cell types present in most tissue sections offers internal negative control sites. (This should be verified by the user.) If staining occurs in the negative control tissue, results with the patient specimen should be considered invalid. C. Negative Control Reagent Use a negative control reagent in place of the primary antibody with a section of each patient specimen to evaluate nonspecific staining and allow better interpretation of specific staining at the antigen site. Ideally, a negative control reagent contains antibody which exhibits no specific reactivity with human tissues (non-human reactive) in the same matrix/solution as the primary antibody. The non-human reactive antibody should be the same subclass and animal species as the primary antibody, diluted to the same immunoglobulin or protein concentration as the primary antibody using the identical diluent. Depending on the type of primary antibody/antiserum used, normal/non-immune serum from the same species as the primary at a protein concentration equivalent to the diluted primary in the same diluent may be suitable for use. Refer to the product insert of each primary antibody and the examples below for specific recommendations. Diluent alone may be used as a less than ideal alternative to the previously described negative control reagents. The incubation period for the negative control reagent should correspond to that of the primary antibody/antiserum. EXAMPLES OF NEGATIVE CONTROL REAGENTS FOR CONCENTRATED ANTIBODIES: Primary Antibody Type Suggested Negative Control Reagent monoclonal mouse non-human reactive monoclonal mouse antibody, produced in antibody, produced in ascites; or ascites, subclass normal/nonimmune mouse serum, specific DAKO Code No. X0910 monoclonal mouse non-human reactive monoclonal mouse antibody produced in antibody produced in tissue culture: tissue culture, subclass DAKO Code Nos. X0931 (IgG 1 ), X0943 specific (IgG 2a ), X0944 (IgG 2b ) or X0942 (IgM); or fetal calf serum* polyclonal rabbit normal/non-immune rabbit serum, antibody, immuno- immunoglobulin fraction, globulin fraction DAKO Code No. X0903 polyclonal rabbit normal/non-immune whole rabbit serum, antibody, whole serum DAKO Code No. X0902 *Fetal calf serum is suitable for use if it is retained in the primary antibody after processing. If panels of antibodies are used on serial tissue sections, the negatively staining areas of one slide may serve as a negative/ nonspecific binding background control for other antibodies.

14 Note: When using DAKO N-Series ready-to-use primary antibodies, use the provided negative control reagent. To differentiate endogenous enzyme activity or nonspecific binding of enzymes from specific immunoreactivity, additional patient tissues may be stained exclusively with prepared substrate chromogen, or prepared streptavidin and substrate chromogen, respectively. For remedial solutions to aberrant test results refer to Troubleshooting, Section 14. D. Assay Verification Prior to initial use of an antibody or staining system in a diagnostic procedure the user should verify the antibody s specificity by testing it on a series of in-house tissues with known positive and negative immunohistochemical performance characteristics. Refer to the quality control procedures outlined in subsections 10.A, 10.B and 10.C; the Quality Control Section of each primary antibody specification sheet and the quality control requirements of the CAP Certification Program for Immunohistochemistry. These quality control procedures should be repeated for each new antibody lot or whenever there is a change in assay parameters. 11. INTERPRETATION OF STAINING A. Positive Control Tissue The positive control tissue should be examined first to ascertain that all reagents are functioning properly. Presence of a red-colored end-product at the site of the target antigen is indicative of positive reactivity. If the positive control tissues fail to demonstrate positive staining, results with the test specimens should be considered invalid. B. Negative Control Tissue The negative control tissue should be examined after the positive control tissue to verify the specific labeling of the target antigen by the primary antibody. The absence of specific staining in the negative control tissue confirms the lack of antibody cross-reactivity to cells/cellular components. If specific staining occurs in the negative control tissue, results with the patient specimen should be considered invalid. Nonspecific staining, if present, will be of diffuse appearance. Sporadic light staining of connective tissue may be observed in sections from excessively formalin fixed tissues. Use intact cells for interpretation of staining results. Necrotic or degenerated cells often stain nonspecifically. C. Patient Tissue Patient tissues stained with the primary antibody should be examined last. Positive staining intensity should be assessed within the context of any non-specific background staining of the negative control reagent. The absence of a specific positive staining reaction can be interpreted as no antigen detected. If necessary, use a panel of antibodies to identify false-negative reactions. D. General Comments Precipitates may form if specimens are allowed to dry during the staining procedure. This may be apparent at the edge of the specimen. Scan tissues at 40x magnification to avoid misinterpretation of results. Depending on the incubation length and potency of the hematoxylin used, counterstaining will result in a pale to dark blue coloration of cell nuclei. Excessive or incomplete counterstaining may compromise proper interpretation of results. 12. GENERAL LIMITATIONS Immunohistochemistry is a multistep diagnostic process that requires specialized training in the selection of the appropriate

15 reagents; tissue selection, fixation, and processing; preparation of the IHC slide; and interpretation of the staining results. Tissue staining is dependent on the proper handling and processing of tissues prior to staining. Improper fixation, freezing, thawing, washing, drying, heating, sectioning or contamination with other tissues or fluids may produce artifacts, antibody trapping, or false-negative results. Inconsistent results may be due to variations in fixation and embedding methods, or to inherent irregularities within the tissue. Excessive or incomplete counterstaining may compromise proper results. Use of old or unbuffered fixatives, or exposure of tissues to excessive heat (greater than 60 C) during processing may result in decreased staining sensitivity. Normal/non-immune sera from the same animal source as the secondary antisera used in blocking steps may cause falsenegative or false-positive results due to auto-antibodies or natural antibodies. False-positive results may be seen due to non-immunologic binding of reagents to tissue sections. 9,15 In some cases the application of an alternate blocking reagent prior to incubation with the primary antibody may be useful for reducing background. A recommended blocking reagent is DAKO Protein Block Serum-Free (Code No. X0909). Unexpected negative reactions in poorly differentiated neoplasms may be due to loss or marked decrease of antigen expression or nonsense mutation in the gene(s) coding for the antigen. Unexpected positive staining in tumors may be from expression of an antigen not usually expressed in morphologically similar normal cells, or from persistence or acquisition of an antigen in a neoplasm that develops morphologic and immunohistochemical features associated with another cell lineage (divergent differentiation). Histopathologic classification of tumors is not an exact science and some literature reports of unexpected staining may be controversial. The clinical interpretation of any positive staining or its absence should be complemented by morphological and histological studies with proper controls. Evaluations should be made within the context of the patient s clinical history and other diagnostic tests. It is the responsibility of a qualified pathologist who is familiar with the antibodies, reagents and methods used to interpret the stained preparation. Staining must be performed in a certified licensed laboratory under the supervision of a pathologist who is responsible for reviewing the stained slides and assuring the adequacy of positive and negative controls. 13. PRODUCT SPECIFIC LIMITATIONS DAKO Corporation provides LSAB 2 System reagents at optimal dilution for use following the provided instructions for IHC on Paraffin-Embedded tissue sections, cryostats and blood smears. Any deviation from recommended test procedures may invalidate declared expected results; appropriate controls must be employed and documented. Users who deviate from recommended test procedures must accept responsibility for interpretation of patient results under these circumstances. Endogenous avidin-binding activity (EABA) has been noted in frozen sections of liver (entire hepatic nodule) and kidney (tubular epithelium), as well as in frozen and formalin-fixed lymphoid tissues (paracortical histiocytes). 5-8 EABA can be suppressed by sequential 20-minute incubations, first with 0.1% avidin and then with 0.01% biotin in 0.05 M Tris-HCl buffer, ph , prior to Section 9.B. Step 1 or use DAKO Biotin Blocking System (Code No. X0590). Endogenous peroxidase or pseudoperoxidase activity can be found in hemoproteins such as hemoglobin, myoglobin, cytochrome, and catalase as well as in eosinophils. 10,11 In

16 formalin-fixed tissue this activity can be inhibited by incubating specimens with 3% hydrogen peroxide for five minutes prior to application of the primary antibody. Blood and bone marrow smears and frozen tissues can be treated with DAKO Peroxidase Blocking Reagent (Code No. S2001). However, this procedure does not abolish the reddish-brown pigment of hemoproteins. Alternatively, a solution of methanol-hydrogen peroxide can be used. Some antigens may become denatured with this procedure. Reagents may demonstrate unexpected reactions in previously untested tissues. The possibility of unexpected reactions in tested tissue groups cannot be completely eliminated due to biological variability of antigen expression in neoplasms, or other pathological tissues. 2 0 Contact DAKO Corporation Technical Services Department at 800/ with documented unexpected reactions. Tissues from persons infected with hepatitis B virus and containing hepatitis B surface antigen (HBsAg) may exhibit nonspecific staining with horseradish peroxidase. 21

17

18 15. REFERENCES 1.Farr AG and Nakane PK. Immunohistochemistry with enzyme labelled antibodies. J Immunol Meth 1981; 47:129 2.Hsu SM, et al. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabelled antibody (PAP) procedures. J Histochem Cytochem 1981; 29:577 3.Guesdon JL, et al. The use of avidin-biotin interaction in immunoenzymatic techniques. J Histochem Cytochem 1979; 27: Giorno R. A comparison of two immunoperoxidase staining methods based on the avidin-biotin interaction. Diag Immunol 1984; 2:161 5.Kiernan JA. Histological and histochemical methods: Theory and practice. New York: Pergamon Press 1981:81 6.Naish SJ (ed). Handbook: Immunochemical staining methods. Carpinteria: DAKO Corporation Wood GS and Warnke R. Suppression of endogenous avidin-binding activity in tissues and its relevance to biotin-avidin detection systems. J Histochem Cytochem 1981; 29: Banerjee D and Pettit S. Endogenous avidin-binding activity in human lymphoid tissue. J Clin Pathol 1984; 37:223 9.Elias JM. Immunohistopathology: A practical approach to diagnosis. Chicago: American Society of Clinical Pathologists Press 1990:46 10.Department of Health, Education and Welfare, National Institute for Occupational Safety and Health. Procedures for the decontamination of plumbing systems containing copper and/or lead azide. Rockville, MD Center for Disease Control Manual Guide Safety Management, No. CDC-22. Decontamination of laboratory sink drains to remove azide salts. Atlanta, Georgia. April 30, National Committee for Clinical Laboratory Standards. Protection of laboratory workers from infectious disease transmitted by blood and tissues: tentative guideline. Villanova, PA 1991; 7(9):Order code M29-P 13.Clinical Laboratory Improvement Amendments of 1988: Final rule, 57FR7163. February 18, Carson, FL (ed). Histotechnology: A Self-Instructional Text. Chicago: ASCP Press 1990:22 15.Larsson L. Tissue preparation methods for light microscopic immunohistochemistry. Applied Immuno 1993;1(1):2 16.Nadji M and Morales AR. Immunoperoxidase: I. The technique and its pitfalls. Lab Med 1983; 14: Sheehan DC and Hrapchack BB. Theory and Practice of Histotechnology. St. Louis: C.V. Mosby Co Elias JM, et al. Special report: Quality control in immunohistochemistry. Amer J Clin Pathol 1989; 92: National Committee for Clinical Laboratory Standards. Internal quality control testing: Principles and definitions; approved guideline. Villanova, PA. 1991; 4: Order code C24-A 20.Herman GE and Elfont EA. The taming of immunohistochemistry: The new era of quality control. Biotech & Histochem 1991; 66: Omata M, et al. Nonimmunologic binding of horseradish peroxidase to hepatitis B surface antigen: A possible source of error in immunohistochemistry. Am J Path 1980; 7 3 : 6 2 6

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