CSA II Biotin-free Tyramide Signal Amplification System. Code K1497 For use with mouse primary antibodies. Limited use license

CSA II Biotin-free Tyramide Signal Amplification System Code K1497 For use with mouse primary antibodies Limited use license This system embodies technology developed by and licensed from NEN Life Science Products, Inc. U.S. Patent No. 5,196,306. This product is distributed and sold to the End-User pursuant to a license from NEN LIFE SCIENCE PRODUCTS, INC. for use by the End-User in manual or automated processing on DakoCytomation instrumentation only of glass microscope slides or other support material (other than microarrays and bio-chips) containing cell or tissue specimens for examining those specimens under a microscope (including automated image capture and analysis systems) for the purpose of detecting target nucleic acids or proteins. Purchase does not include or carry any right to resell or transfer this product either as a stand alone product or as a component of another product. Any use of this product other than the licensed use without the express written authorization of NEN LIFE SCIENCE PRODUCTS, INC. is strictly prohibited. Intended use For In Vitro diagnostic use. These instructions apply to the CSA II (code K1497). CSA II is a biotin-free tyramide signal amplification system for immunohistochemistry. This system is intended for use with primary antibodies from mouse supplied by the user for the qualitative identification of antigens by light microscopy in normal and pathological formalin-fixed, paraffin-embedded tissues. This system may be used with manual procedures and/or with DakoCytomation instrumentation. Refer to the General Instructions for Immunohistochemical Staining of IHC procedures for: (1) Principle of Procedure, (2) Materials Required, Not Supplied, (3) Storage, (4) Specimen Preparation, (5) Staining Procedure, (6) Quality Control, (7) Troubleshooting, (8) Interpretation of Staining, (9) General Limitations. Summary and explanation Principles of procedure The CSA II System is a highly sensitive immunohistochemical (IHC) staining procedure incorporating a signal amplification method based on the peroxidase-catalyzed deposition of a fluorescein-labelled phenolic compound, followed by a secondary reaction with a peroxidase-conjugated anti-fluorescein. 1-5 In the procedure, a mouse primary antibody is first detected with a peroxidase-conjugated secondary antibody. The next step utilizes the bound peroxidase to catalyze oxidation of a fluorescein-conjugated phenol (fluorescyl-tyramide) which then precipitates onto the specimen. The procedure is continued with detection of the bound fluorescein by a peroxidase-conjugated anti-fluorescein. Staining is completed using diaminobenzidine/hydrogen peroxide as chromogen/substrate, and can be observed with a light microscope. In comparison to standard immunohistochemical methods, such as labelled streptavidin biotin (LSAB) or avidin-biotin complexes (ABC), tyramide amplification methods have been reported to be many fold more sensitive. 3,4 The CSA II System is a simplified version of the extremely sensitive Catalyzed Signal Amplification System (code K1500) that utilizes biotinyl-tyramide. The highly sensitive CSA II System allows for the detection of very small quantities of target protein, as well as for the use of low affinity antibodies. This reagent system utilizes fluorescyl-tyramide, rather than biotinyl-tyramide, and does not contain avidin/biotin reagents, thus eliminating potential background staining due to reactivity with endogenous biotin. 6,7 The specimens are first incubated with Peroxidase Block for five minutes to quench endogenous peroxidase activity. The specimens are then incubated for five minutes with a protein block to suppress nonspecific binding of subsequent reagents, followed by a 15-minute incubation with an appropriately characterized and diluted mouse primary antibody or negative control reagent (user provided). This is followed by sequential 15-minute incubations with anti-mouse immunoglobulins-hrp, fluorescyl-tyramide hydrogen peroxide (amplification reagent) and anti-fluorescein-hrp. Staining is completed by a five-minute incubation with 3,3' diaminobenzidine tetrahydrochloride (DAB)/hydrogen peroxide, which results in a brown precipitate at the antigen site. (106817-003) 302800EFG_042004 p. 1/10

Reagent provided Code K1497 The following materials are included in this kit: Quantity 1x15 ml Description Peroxidase Block 3% hydrogen peroxide in water. 1x15 ml Protein Block Serum-free protein in buffer with 0.015 mol/l sodium azide. 1x15 ml Anti-Mouse Immunoglobulins-HRP Anti-mouse immunoglobulins conjugated to horseradish peroxidase in buffer containing stabilizing protein and an antimicrobial agent. 1x15 ml Amplification Reagent Fluorescyl-tyramide and hydrogen peroxide in buffer containing stabilizing protein and an antimicrobial agent. 1x15 ml Anti-Fluorescein-HRP Anti-fluorescein antibody conjugated to horseradish peroxidase in buffer containing stabilizing protein and an antimicrobial agent. 1x18 ml Buffered Substrate Buffer containing hydrogen peroxide and a preservative. 1x1 ml Liquid DAB Chromogen 3,3'-diaminobenzidine chromogen solution. Materials required, but not supplied Antibody diluent, such as Antibody Diluent with Background Reducing Components (code S3022) Control specimens, positive and negative Counterstain, such as Lillie s Modified Mayer s Hematoxylin (code S3301 for use on the Dako Autostainer or code S3302 for Manual Staining) Light microscope Mounting media such as Glycergel Mounting Medium (code C0563), Faramount, Aqueous Mounting Medium, Ready-to-use (code S3025), or a nonaqueous permanent mounting medium, such as Permanent Mounting Media (code S3026) Mouse primary antibodies and negative control reagents Wash buffer, such as TBST (Tris Buffered Saline With Tween), 10x Concentrate (code S3306) Automated Staining Dako Autostainer (code S3400) or Autostainer Plus (code S3800). Refer to the Dako Autostainer User Guide for necessary components Optional materials required, but not supplied Refer to primary antibody specification sheet Target retrieval solution, modified citrate buffer (ph 6.1), such as Target Retrieval Solution, Ready-to-use (code S1700) or 10x Concentrate (code S1699) Water bath (capable of maintaining 95 99 C), steamer, pressure cooker or autoclave for performing target retrieval (106817-003) 302800EFG_042004 p. 2/10

Precautions Product Specific 1. For professional users. 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 NaN 3 may react with lead and copper plumbing to form highly explosive metal azides. Upon disposal, flush with large volumes of water to prevent azide build-up in plumbing. 8,24 3. Minimize microbial contamination of reagents or increase in nonspecific staining may occur. 4. As with any product derived from biological sources, proper handling procedures should be used. 5. Wear appropriate Personal Protective Equipment to avoid contact with eyes and skin. 6. Unused solution should be disposed of according to local, State and Federal regulations. 7. Safety Data Sheet available for professional users on request. General 1. Do not use reagents beyond expiration date. 2. Do not substitute reagents from kits of other manufacturers. 3. Do not store system components or perform staining in strong light, such as direct sunlight. Note: Slides must be protected from light during the Amplification Reagent incubation step. Risk and Safety Statements Dab Chromogen R 40 Limited evidence of a carcinogenic effect. R43 May cause sensitization by skin contact. R68 Possible risk of irreversible effects. S35 This material and its container must be disposed of in a safe way. S 36/37 Wear suitable protective clothing and gloves. Storage Reagents of CSA II System are to be stored at 2 8 C. Do not freeze. Do not use after expiration printed on reagent vials and kit label. 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 DakoCytomation Technical Support. Reagent preparation Wash Buffer Investigation has shown that 0.05 mol/l Tris-HCl, ph 7.6, containing 0.3 mol/l NaCl and 0.1% Tween 20, without sodium azide, significantly aids in minimizing background staining with this system. A recommended wash buffer is TBST 10x (code S3306). Wash buffers containing sodium azide inactivate horseradish peroxidase and result in negative staining. Store unused buffer at 2 8 C. Discard buffer if cloudy in appearance. Automated Staining Automation Buffer, 10x (code S3006) can be used for the general wash buffer rinses on the Dako Autostainer for the CSA II automated protocol, but a 5 10 minute auxiliary wash with TBST (code S3306) should be used after the hydrogen peroxide, the primary antibody and the anti-mouse-hrp reagents. Distilled or deionized water may be used for rinsing after the substrate-chromogen and counterstain steps. Manual Staining Tissue sections should be washed in up to three fresh baths of TBST (code S3306) for 3 5 minutes between each step of the CSA II protocol. DAB Substrate-Chromogen Preparation One ml of the substrate-chromogen is sufficient for up to ten tissue sections. STEP 1 STEP 2 Depending on the number of slides to be stained, transfer enough 1 ml aliquots of buffer from the Buffered Substrate bottle into a test tube. For each 1 ml of Buffered Substrate, add 1 drop (20 µl) from the Liquid DAB Chromogen bottle. Mix immediately. The prepared substrate-chromogen is stable approximately five days when stored at 2 8 C. This reagent should be mixed thoroughly prior to use. Any precipitate developing in the reagent does not affect staining quality. Dilution of Concentrated Primary Antibody and Negative Control Dilute primary antibody using a diluent containing stabilizing protein. Use of Antibody Diluent with Background Reducing Components (code S3022) is highly recommended for minimizing background staining. An appropriate negative control reagent consists of mouse immunoglobulin of the same isotype as the primary antibody diluted to an immunoglobulin concentration equivalent to that of the diluted primary antibody. (106817-003) 302800EFG_042004 p. 3/10

Specimen preparation Paraffin-embedded 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 in the General Instructions for Immunohistochemical Staining. These are guidelines only. Optimal procedures must be determined and verified by the user. 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. 10-15 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. After fixation, 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 in a cool place (15 22 C). Mounting of Tissue Sections Sectioned tissues cut from paraffin-embedded blocks are collected on clean glass slides. Tap slides to remove as much water as possible. Water droplets remaining on the tissue during the drying process may adversely affect tissue staining. Dry in an oven for one to two hours at 60 C or less or allow to dry at room temperature for 15 24 hours. For increased adhesion of tissue sections during IHC staining procedures, use of poly-l-lysine coated slides, charged slides or Silanized Slides (code S3003) is suggested. Use of Silanized or charged slides is strongly recommended for staining procedures requiring target retrieval. Automated and manual staining procedure Procedural Notes The user should read these instructions carefully and become familiar with the kit contents prior to use. All reagents should be equilibrated to room temperature (20 25 C) prior to immunostaining; likewise, all incubations are optimized for performance at room temperature. The reagents and instructions supplied in this kit have been designed for optimal performance. Further dilution of the ready-to-use reagents may give erroneous results. Note: For Automated Staining, the tinted Dako Autostainer cover should be closed during the Amplification Reagent step for optimal staining performance. For Manual Staining, protect slides from light during the incubation of the Amplification Reagent. Do not allow tissue sections to dry during the staining procedure. Dried tissue sections may display nonspecific staining. Cover slides exposed to drafts. If prolonged incubations are used, place slides in a humid environment. For reduction of nonspecific background staining it is recommended that tissue sections be rinsed with TBST according to the wash buffer recommendations in Reagent Preparation section. Deparaffinization of Tissue Sections Prior to staining, tissue slides must be deparaffinized to remove embedding medium and then rehydrated. Avoid incomplete removal of paraffin. Residual embedding medium will result in increased nonspecific staining. Proteolytic Digestion and Target Retrieval Formaldehyde is known to induce conformational changes in the antigen molecules by forming intermolecular cross-linkages. Excessive formalin fixation can mask antigenic sites and diminish specific staining. In standard IHC procedures, these sites may be revealed with proteolytic digestion of tissue prior to immunostaining. Note: Use of proteolytic digestion may result in high background staining with the CSA II System and is therefore not recommended. Target retrieval using heat is the preferred alternative to proteolytic digestion, resulting in a slight increase in background staining concurrent with a significant increase in specific staining with many primary antibodies. To determine if target retrieval treatment of tissues is warranted, see the specification sheet provided with the primary antibody; for some primary antibodies this procedure is required. The recommended target retrieval procedure involves immersion of tissue sections mounted on slides in a modified citrate buffer (ph 6.1) such as Target Retrieval Solution, Ready-to-use (code S1700), or 10x Concentrate (code S1699); refer to package insert for complete instructions and heating prior to IHC staining. Rinse thoroughly with wash buffer solution and continue with the staining procedure. Note: Increased background staining may result from the use of a high ph (ph 10) target retrieval solution with CSA II System and is therefore not recommended. 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Automated Staining Procedure STEP 1 Select Protocol Template Auto Program STEP 2 Place reagents and buffer in the Dako Autostainer reagent rack STEP 3 Load the slides on to the Dako Autostainer STEP 4 Start the run STEP 5 Remove slides from the Dako Autostainer STEP 6 Proceed with mounting Note: Counterstaining is included in the recommended programming grid. Counterstaining can be run manually on the lab bench as an alternative option. Manual Staining Procedure STEP 1 PEROXIDASE BLOCK Tap off excess liquid. Using a lintless tissue, carefully wipe around the specimen to remove any remaining liquid and to keep reagent within the prescribed area. Apply enough Hydrogen Peroxide to cover specimen. Incubate 5 (±1) minutes. Rinse gently with distilled water or wash buffer solution from a wash bottle (do not focus flow directly on tissue) and place in a fresh wash buffer bath. STEP 2 STEP 3 STEP 4 STEP 5 STEP 6 STEP 7 PROTEIN BLOCK Tap off excess liquid and wipe slide as before. Apply enough Protein Block to cover specimen. Incubate 5 (±1) minutes. Do not rinse off protein block. PRIMARY ANTIBODY OR NEGATIVE CONTROL REAGENT Tap off excess Protein Block and wipe slide as before. Apply enough user-prepared primary antibody or negative control reagent to cover specimen. Incubate 15 (±1) minutes unless otherwise specified in primary antibody specification sheet. Rinse gently with wash buffer solution from a wash bottle and place in up to three fresh TBST buffer baths for 3 5 minutes each. ANTI-MOUSE IMMUNOGLOBULINS-HRP Wipe slide as before. Apply enough Anti-Mouse Immunoglobulins-HRP to cover specimen. Incubate 15 (±1) minutes. Rinse slide as in Step 3. Place in up to three fresh wash buffer baths for 3 5 minutes each. AMPLIFICATION REAGENT Note: Slides must be protected from light during this incubation step for optimal staining. Wipe slide as before. Apply enough Amplification Reagent to cover specimen. Incubate 15 (±1) minutes protected from light. Rinse slide as before. Place in up to three fresh wash buffer baths for 3 5 minutes each. ANTI-FLUORESCEIN-HRP Wipe slide as before. Apply enough Anti-Fluorescein-HRP to cover specimen. Incubate 15 (±1) minutes. Rinse slide as before. Place in up to three fresh wash buffer baths for 3 5 minutes each. LIQUID DAB SUBSTRATE-CHROMOGEN Wipe slide as before. Apply enough prepared Liquid DAB Substrate-Chromogen to cover specimen. Incubate 5 (±1) minutes. Rinse with distilled or deionized water. (106817-003) 302800EFG_042004 p. 5/10

STEP 8 PROCEED WITH COUNTERSTAIN AND MOUNTING Dako Autostainer Programming Grid for CSA II The programming grid above is a DakoCytomation Protocol Template containing protocol elements to run CSA II. This example includes the recommended reagents and incubation times for an optimal CSA II run for the Autostainer (code S3004) and Autostainer Plus (code S3800). Set up a protocol template and Auto Program with these recommended steps and incubation times. The primary antibody and pretreatment has been inserted as an example. Application of Target Retrieval is antibody specific and may not be required. 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. See references 16 through 18 for additional quality control information. Positive Control Tissue Known positive control tissues should be utilized for monitoring the correct performance of processed tissues and test reagents. If the positive control tissues fail to demonstrate positive staining, results with the test specimens should be considered invalid. Negative Control Reagent Use a negative control reagent in place of the primary antibody with a section of each test specimen to evaluate nonspecific staining and to allow better interpretation of specific staining at the antigen site. 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. Refer to the General Instructions for Immunohistochemical Staining for further information on positive and negative controls. Staining interpretation The positive control specimens should be examined first to ascertain that all reagents are functioning properly. The presence of a brown-colored end product at the site of the target antigen is indicative of positive reactivity. If the positive control specimens fail to demonstrate positive staining, results with the test specimens should be considered invalid. The negative control specimens should be examined to verify the specific labelling of the target antigen by the primary antibody. The absence of specific staining in the negative control specimens confirms the specificity of the primary antibody. Nonspecific staining, if present, will be of dot-like or diffuse appearance. The high sensitivity of this system is due to its great level of signal amplification; any nonspecific background staining present will also be amplified. For this reason, use of negative control reagents on a section of each control and test specimen is recommended as an aid in interpretation of staining (see Limitations for more discussion on nonspecific staining). Sporadic light staining of connective tissue may be observed in sections from excessively formalin-fixed tissues. Use intact cells for the interpretation of staining results. Necrotic or degenerated cells often stain nonspecifically. False-positive results may be seen due to non-immunologic binding of reagents to tissue sections. (106817-003) 302800EFG_042004 p. 6/10

Limitations For optimal staining intensity, protect slides from light during the Amplification Reagent incubation (Step 5). Use of proteolytic digestion may result in high background staining with the CSA II System and is therefore not recommended. For reduction of nonspecific background staining it is recommended that tissue sections be rinsed with wash buffer (see Reagent Preparation) and incubated at room temperature in up to three fresh baths of wash buffer for three to five minutes each between each reagent step in the CSA II System protocol. Endogenous immunoglobulin, present in human tissue specimens, may exhibit various levels of cross-reactivity with secondary anti-immunoglobulin antibodies. The anti-mouse immunoglobulin-hrp secondary antibody used in this system has been absorbed with normal human serum to reduce cross-reactivity. However, due to the high sensitivity of this system, cross-reactivity to endogenous immunoglobulin may still be detected in some specimens. Background staining due to endogenous immunoglobulin is best recognized as staining in the negative control around blood and lymph vessels, in connective tissue, and in extravascular tissue spaces. Certain types of epithelium, particularly those associated with the digestive tract, may also display endogenous immunoglobulin. Staining due to endogenous immunoglobulin may also be increased by using target retrieval methods. For this reason, use of appropriate negative control reagents and tissues are required for interpreting any positive staining where presence of endogenous immunoglobulin is suspected. Endogenous peroxidase or pseudoperoxidase activity can be found in hemoproteins such as hemoglobin, myoglobin, cytochrome, and catalase, as well as in certain leucocytes. 19,20 This activity can be eliminated by incubating specimens with Peroxidase Block for 5 minutes (Step 1 of the CSA II System protocol) prior to the application of the primary antibody. Tissues containing hepatitis B surface antigen (HBsAg) may exhibit nonspecific staining with horseradish peroxidase. 21 Automated troubleshooting Problem Probable Cause Suggested Action 1. No staining of slides. 1a. Programming error. 1a. Review application of reagents. Reagents not used in proper order. 1b. Reagent vials were not loaded in the correct locations in the reagent racks. 1b. Check the Reagent Map to verify the proper location of reagent vials. 2. Weak staining of slides. 3. Excessive background staining of slides. 1c. Insufficient reagent in reagent vial. 1c. Ensure that enough reagent is loaded into the reagent vials prior to beginning the run. Refer to Reagent Map for volumes required. 1d. Sodium azide in buffer bath. 1d. Use fresh, azide-free buffer. 2a. Slides not incubated long 2a. Review recommended incubation times. enough with antibodies or substrate. 2b. Antigen under investigation requires target retrieval. 2c. Specimens exposed to light during Amplification Reagent incubation. 2d. Antibody concentration too low. 3a. Specimens contain high levels of endogenous immunoglobulin. 3b. Specimens contain high endogenous peroxidase activity. 3c. Paraffin incompletely removed. 2b. Perform target retrieval. 2c. Protect specimens from light during Amplification Reagent incubation. 2d. Re-titer antibody. 3a. Compare test tissue with the negative control tissue for differences. 3b. Use longer incubation time of hydrogen peroxide (Step 1). 3c. Use fresh xylene or toluene baths. If several slides are stained simultaneously, the second xylene bath should contain fresh xylene. 3d. Slides not thoroughly rinsed. 3d. Ensure that the Dako Autostainer is properly primed prior to running. Check to make sure that adequate buffer is provided for entire run. Include auxiliary washes with TBST (code S3306) as specified. (106817-003) 302800EFG_042004 p. 7/10

4. Tissue detached from slides. 3e. Sections dried during staining. 3f. Slides dried while loading the Dako Autostainer. 3g. Nonspecific binding of reagents to tissue section. 3h. Nonspecific binding of primary antibody to specimen. 3i. High ph Target Retrieval Solution used for heat retrieval prior to staining. 3j. Proteolytic digestion used 3e. Verify that the appropriate volume of reagent is being applied to slides. Make sure the Dako Autostainer is run with the hood in the closed position and that it is not exposed to excessive heat or drafts. 3f. Ensure slides remain wet with buffer while loading and prior to initiating the run. 3g. Use longer incubation time of Protein Block (Step 2). 3h. Dilute primary antibody in diluent containing carrier protein. Antibody Diluent with Background Reducing Components (code S3022) is recommended. 3i. Use a modified citrate buffer (ph 6.1) for target retrieval. 3j. Avoid use of proteolytic digestion with this prior to staining. staining system. 4a. Use of incorrect slides. 4a. Use charged (SuperFrost Plus) or Silanized slides such as DakoCytomation s Silanized Slides (code S3003). Manual troubleshooting Problem Probable Cause Suggested Action 1. No staining of slides. 1a. Reagents not used in proper 1a. Review application of reagents. order. 1b. Sodium azide in buffer bath. 1b. Use fresh, azide-free buffer. 2. Weak staining of slides. 2a. Sections retain too much solution after wash bath. 2a. Gently tap off excess solution before wiping around slides. 2b. Slides not incubated long 2b. Review recommended incubation times. enough with antibodies or substrate. 2c. Antigen under investigation 2c. Perform target retrieval. requires target retrieval. 2d. Specimens exposed to light during Amplification Reagent 2d. Protect specimens from light during Amplification Reagent incubation. incubation. 2e. Antibody concentration too 2e. Re-titer antibody. 3. Excessive background staining of slides. low. 3a. Specimens contain high levels of endogenous immunoglobulin. 3b. Specimens contain high endogenous peroxidase activity. 3c. Paraffin incompletely removed. 3a. Compare test tissue with the negative control tissue for differences. 3b. Use longer incubation time of hydrogen peroxide (Step 1). 3c. Use fresh xylene or toluene baths. If several slides are stained simultaneously, the second xylene bath should contain fresh xylene. 3d. Slides not thoroughly rinsed. 3d. Use fresh solutions in buffer baths and wash bottles. TBST is recommended. Incubation sections in up to three fresh TBST baths for 3 5 minutes each between protocol steps. 3e. Faster than normal substratechromogen reaction due to excessively high room temperature. 3f. Sections dried during staining procedure. 3g. Nonspecific binding of reagents to tissue section. 3h. Nonspecific binding of primary antibody to specimen. 3e. Use shorter incubation time with substrate-chromogen solution. 3f. Use humidity chamber. Wipe only 3 to 4 slides at a time before applying reagent. 3g. Use longer incubation time of Protein Block (Step 2). 3h. Dilute primary antibody in diluent containing stabilizing protein. Antibody Diluent with Background Reducing Components (code S3022) is recommended. (106817-003) 302800EFG_042004 p. 8/10

4. Tissue detached from slides. 5. Run-to-run variability of staining intensity. 3i. High ph Target Retrieval Solution used for heat Retrieval prior to staining. 3j. Proteolytic digestion used 3i. Use a modified citrate buffer (ph 6.1) for target retrieval. 3j. Avoid use of proteolytic digestion with this prior to staining. staining system. 4a. Use of incorrect slides. 4a. Use charged (SuperFrost Plus) or Silanized slides such as DakoCytomation s Silanized Slides (code S3003). 5a. Variations in room temperature. 5b. Slight variations in length of reagent incubations. 5c. Variations in number and incubation times of wash buffer baths. 5a. Regulate room temperature to narrow range. 5b. Time incubations consistently. 5c. Follow identical protocol run-to-run. References 1. Bobrow MN, Harris TD, Shaughnessy KJ, Litt GJ. Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays. J Immunol Methods 1989; 125:279 2. Bobrow MN, Shaughnessy KJ, Litt GJ. Catalyzed reporter deposition, a novel method of signal amplification. II. Application to membrane immunoassays. J Immunol Methods 1991; 137:103 3. Van Gijlswijk RPM, Zijlmans HJMAA, Wiegant J, Bobrow MN, Erickson TJ, Adler KE, Tanke HJ, Raap AK. Fluorochrome-labeled tyramides: use in immunocytochemistry and fluorescence in situ hybridization. J Histochem Cytochem 1997; 45(3):375 4. Von Wasielewski R, Mengel M, Gignac S, Wilkens L, Werner M, Georgii A. Tyramine amplification technique in routine immunohistochemistry. J Histochem Cytochem 1997; 45(11):1455 5. Bobrow MN, Litt GJ, Shaughnessy KJ, Mayer PC, Conlon J. The use of catalyzed reporter deposition as a means of signal amplification in a variety of formats. J Immunol Methods 1992; 150:145 6. Wood GS and Warnke R. Suppression of endogenous avidin-biotin activity in tissues and its relevance to biotin-avidin detection systems. J Histochem Cytochem 1981; 29(10):1196 7. Banerjee D and Pettit S. Endogenous avidin-binding activity in human lymphoid tissue. J Clin Pathol 1984; 37:223 8. Department of Health, Education and Welfare, National Institute for Occupational Safety and Health, Rockville, MD. Procedures for the decontamination of plumbing systems containing copper and/or lead azides. DHHS (NIOSH) Publ. No. 78-127, Current 13. August 16, 1976 9. National Committee for Clinical Laboratory Standards. Protection of laboratory workers from instrument biohazards and infectious disease transmitted by blood, body fluids and tissue; approved guideline. Villanova, PA. 1997; Order Code M29-A 10. Kiernan JA. Histological and histochemical methods: Theory and practice. New York: Pergamon Press 1981; 81 11. Boenisch T. In: Naish SJ, ed. Handbook immunochemical staining methods. Carpinteria: DakoCytomation 1989 12. Nadji M and Morales AR. Immunoperoxidase: I. The technique and its pitfalls. Lab Med 1983; 14:767 13. Banks PM. Diagnostic applications of an immunoperoxidase method in hematopathology. J Histochem Cytochem 1979; 27:1192 14. Culling CFA, Reid PE, Sinnott NM. The effect of various fixatives and trypsin digestion upon the staining of routine paraffin-embedded sections by the peroxidase-antiperoxidase and immunofluorescent technique. J Histotech 1980; 3:10 15. Carson FL (ed). Histotechnology: A self-instructional text. Chicago: ASCP Press 1990:22 16. Elias JM, Gown AM, Nakamura RM, Wilbur DC, Herman GE, Jaffe ES, Battifora H, Brigati DJ. Special report: Quality control in immunohistochemistry. Amer J Clin Pathol 1989; 92:836 17. National Committee for Clinical Laboratory Standard. Internal quality control testing: principles and definitions; approved guideline. Villanova, PA 1991; Order code C24-A:4 18. Herman GE and Elfont EA. The taming of immunohistochemistry: The new era of quality control. Biotech & Histochem 1991; 66:194 19. Escribano LM, Gabriel LC, Villa E, Navarro JL. Endogenous peroxidase activity in human cutaneous and adenoidal mast cells. J Histochem Cytochem 1987; 35:213 20. Elias JM. Immunohistopathology: A practical approach to diagnosis. Chicago: Amer Soc Clin Pathol Press 1990; 46 21. Omata M, Liew C-T, Ashcavai M, Peters RL. Nonimmunologic binding of horseradish peroxidase to hepatitis B surface antigen: A possible source of error in immunohistochemistry. Amer J Pathol 1980; 73:626 22. Tubbs RR, Gephardt GN, Petras RE. Atlas of immunohistology. Chicago: Amer Soc Clin Pathol Press 1986 23. Nadji M and Morales AR. Immunoperoxidase techniques, a practical approach to tumor diagnosis. Chicago: Amer Soc Clin Pathol Press 1986 24. Center for Disease Control Manual Guide Safety Management, No. CDC-22, Atlanta, GA. Decontamination of laboratory sink drains to remove azide salts. April 30, 1976 (106817-003) 302800EFG_042004 p. 9/10

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