Tissue Tackle AEC Mouse Immunohistochemistry System Cat # HCS26

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1 Tissue Tackle AEC Mouse Immunohistochemistry System Cat # HCS26 Table of Contents Page Intended Use... 1 Background... 2 Principle of the Assay... 2 Materials Provided... 3 Materials Required But Not Provided... 3 Precautions and Recommendations... 3 Protocol Summary... 4 Detailed Protocol... 5 Troubleshooting References Reagent Stability... 7 Intended Use The Oncogene Research Products Tissue Tackle Mouse Immunohistochemistry System is specifically designed for optimum immunohistochemical staining using mouse primary antibodies on mouse tissues. This complete kit includes all required pretitrated reagents in precision dropper bottles sufficient for 60 tests. This assay is for research use only and not for use in diagnostic or therapeutic procedures. Upon receipt, store the entire kit at +4 o C until use. Oncogene Research Products Page 1 of 7 Tissue Tackle IHC System

2 Background Immunohistochemical detection of mouse antigens with mouse monoclonal antibodies is frequently compromised by the high backgrounds that are caused by specific interactions of the enzyme-, fluor- or biotin- conjugated anti mouse immunoglobulin (Ig) secondary reagents with endogenous Igs that are present in the rodent tissue sample. This problem can be overcome by addition of unconjugated Fab fragments of anti mouse Ig to cover the endogenous rodent antibodies prior to addition of the primary and secondary antibodies. However, addition of anti mouse Ig antibody to cover the endogenous mouse antibody can be time consuming, and complete masking of the endogenous immunoglobulins can be difficult to achieve. An alternative approach involves direct biotinylation of the primary antibody. The Tissue Tackle Mouse Immunohistochemistry System uses a labeling reagent that contains biotinylated proteins that bind specifically to the Fc region of the primary antibody. This labeling reagent will add multiple biotin residues to a primary antibody even in the presence of contaminating proteins. After addition of the biotinylated primary antibody to the sample, the antigen/biotinylated primary antibody complex is detected by addition of a streptavidin conjugated enzyme and the subsequent addition of enzyme substrate. Because of the strong interaction of streptavidin with biotin, background signals arising from random binding of enzyme linked secondary reagents is minimized. Principle of the Assay Antigens in tissues and cells are detected by a two-stage process: binding of mouse primary antibody to a specific epitope followed by detection of the antigen/primary antibody complex with a colorimetric reaction. Tissues or cell preparations are frozen or fixed, sectioned, and attached to slides. The sections are dewaxed (if paraffin-embedded), treated with a target retrieval solution (if required) and blocked with a proteinaceous blocking solution. The primary antibody is labeled with a biotinylation reagent that specifically labels the F C region of IgG s. After addition of a blocking reagent to stop the biotinylation reaction, the labeled antibody is added to the tissue section. The antigen/biotinylated primary antibody complex is detected by addition of horseradish peroxidase-streptavidin conjugate and AEC substrate. When adequate color development is seen, the slides are washed in water to stop the reaction, counterstained and covered with a mounting medium. With this detection system, signal amplification is achieved through labeling of each primary antibody molecule with multiple biotin residues. The large number of biotin residues per antibody molecule allows for the localization of a relatively large number of enzyme molecules at the site of the target antigen with a consequent enhancement of primary antibody sensitivity. The AEC (Aminoethyl carbazole) substrate offers high sensitivity for light microscopic observations. The bright brick-red dye precipitate produces maximal contrast with blue counterstains and reproduces well by color photomicrography. Materials Provided Peroxide Block: 6 ml ready-to-use. An aqueous solution of hydrogen peroxide and stabilizers. Power Block Reagent: 6 ml ready-to-use. A highly effective universal protein blocking reagent. Contains casein and proprietary additives in PBS with 15 mm sodium azide. Biotin Labeling Reagent 10X: 0.6 ml. Biotinylated mouse immunoglobulin binding proteins in phosphate buffered saline with stabilizers and 15 mm sodium azide. MouseBlock Reagent 10X: 0.6 ml. In phosphate buffered saline with stabilizers and 15 mm sodium azide. HRP-Streptavidin Conjugate: 6 ml ready-to-use. Pretitrated streptavidin-enzyme conjugate in phosphate buffered saline with stabilizers and thimerosal. Oncogene Research Products Page 2 of 7 Tissue Tackle IHC System

3 AEC Single-Reagent Substrate: 6 ml ready-to-use. The "Single-Reagent" formulation of AEC replaces the usual two-or three- component formulations. The brick-red precipitate produced is insoluble in water but is soluble in alcohol and xylene. PBS Tablets: 12 tablets to make 1.2 liters. Tablets are dissolved in deionized water (1 tab/100 ml H 2 O). 137 mm NaCl, 10 mm Na-phosphate buffer ph 7.4, 2.7 mm KCl after reconstitution. Materials Required But Not Provided Tween % Ethanol Mounting Media 70% Ethanol Xylene Hematoxylin Counterstain PBS-Tween is prepared by dissolving the provided 50 PBS tablets in 5000 ml deionized water. The solution may be autoclaved. Add Tween -20 to a final concentration of 0.1%. Precautions and Recommendations Upon receipt, store the entire kit at +4 o C until use. Do not freeze. Allow kit reagents to warm to room temperature before use. Do not expose reagents to excessive light. Wear disposable gloves and eye protection. Do not use the kit beyond the expiration date. Do not mix reagents from different kits. Do not mouth pipette or ingest any of the reagents. The buffers and reagents used in this kit contain anti-microbial and anti-fungal reagents. Care should be taken to prevent direct contact with these products. Do not smoke, eat, or drink when performing the assay or in areas where samples or reagents are handled. Oncogene Research Products Page 3 of 7 Tissue Tackle IHC System

4 Protocol Summary Prepare Labeled-Antibody Mixture De-paraffinize the slide Apply Peroxide Block (5 min) Wash in water for 5 min Add MouseBlock to Labeled-Antibody Mixture from Step 1 Apply Power Block Reagents (3 min) Apply Blocked Labeled Antibody Mixture from step 5 (30 min to overnight) Rinse, wash for 5 min, and rinse with PBS/Tween Apply Peroxidase-Streptavidin (20 min) Rinse, wash for 5 min, and rinse with PBS/Tween Apply AEC Substrate (~10 min) Wash in water Counterstain in Hematoxylin (2 min) Rinse, wash for 5 min, and rinse with PBS/Tween Apply Mounting Medium Dry Oncogene Research Products Page 4 of 7 Tissue Tackle IHC System

5 Detailed Protocol Note: Optimum signal-to-noise ratio is dependent on the antibody dilution, its specificity, and the incubation conditions. Refer to the data sheet of the antibody for optimal conditions. 1. Prepare Labeled Antibody: To the pre-diluted primary antibody (1X) add 1/10 volume (1 volume = volume of primary antibody) of Biotin Labeling Reagent. Mix well and incubate for at least 30 minutes at room temperature or overnight at +4 o C. The labeled primary antibody can be stored up to 6 months at +4 o C. 2. Deparaffinization: Deparaffinize in xylene, using 3 changes, 5 minutes each. Hydrate with 100% ethanol, using 2 changes, 5 minutes each. Hydrate with 95% ethanol, using 2 changes, 5 minutes each. Rinse in distilled water for 5 minutes. 3. Target Retrieval (if required): Place slides in a bath containing Target Retrieval Solution. Place in a microwave and bring to a boil. Lower power to about 20% and simmer for 15 minutes. Let cool for 20 minutes. Wash in running water for 3 minutes. Note: Aldehyde fixation effects may be reversed by target retrieval procedures producing a substantial improvement in immunostaining with most antibodies. Check the datasheet of the antibody for optimal retrieval conditions. 4. Place enough drops of Peroxide Block to cover the section and incubate for 5 minutes at room temperature. Drain and blot by carefully around the section with a paper towel. Wash in running water for 3 minutes. Drain and blot carefully.. Note: The Peroxide Block might damage the morphology of blood smears and peroxidase-rich tissues. For such tissues, inactivate peroxidases by dipping the slides in 0.3% H 2 O 2 in methanol for 10 minutes after step 1, followed by the water wash. 5. To the test tube containing the labeled-antibody mixture from step 1, add 1/10 volume (1 volume = volume of primary antibody in step 1) of MouseBlock Reagent, mix well, and incubate for 3 minutes at room temperature. Use within 15 minutes. The volume of MouseBlock Reagent added in this step should be equal to the volume of Biotin Labeling Reagent added in step 1. After addition of the MouseBlock reagent, the labeled antibody mixture should be used within minutes or a decrease in signal intensity may be seen. 6. Place enough drops of Power Block Reagent to cover the section and incubate for 3 minutes at room temperature. Drain and blot carefully. Note: Antibodies attach non-specifically to highly charged sites; this non-specific binding can be minimized by the use of a proteinaceous blocking reagent 7. Place enough volume of the blocked labeled-antibody mixture from step 8 to cover the section. Incubate as recommended for the primary antibody (usually 30 minutes at room temperature). Drain and blot carefully. Note: optimum signal-to-noise ratio is dependent on the antibody dilution, its specificity, and the incubation conditions. Refer to the data sheet of the antibody for optimum conditions. 8. Rinse slides with PBS-Tween ( PBS with 0.1% Tween -20) from a wash bottle. Transfer slides to a bath containing PBS-Tween and incubate for 5 minutes. Rinse with PBS-Tween -20. Drain and blot carefully. 9. Place enough drops of streptavidin-horseradish Peroxidase Conjugate to cover the section. Incubate for 20 minutes at room temperature. Drain and blot carefully. 10. Rinse slides with PBS-Tween ( PBS with 0.1% Tween -20) from a wash bottle. Transfer slides to a bath containing PBS-Tween and incubate for 5 minutes. Rinse with PBS-Tween -20. Drain and blot carefully. 11. Place enough drops of AEC Substrate Solution to cover the section. Incubate until adequate color development is seen. Note: The color reaction is usually complete within 10 minutes. The HRP enzyme is no longer active after 45 minutes, so incubation beyond 45 minutes is not recommended. 12. Wash in running water for 5 minutes. Drain and blot carefully. Oncogene Research Products Page 5 of 7 Tissue Tackle IHC System

6 13. Place enough drops of Hematoxylin counterstain to cover the section. Incubate for 2 min at RT. Drain and "blue" in alkaline water if required. 14. Wash in water for at least 5 min. Drain and blot carefully. 15. Place enough drops of Mounting Medium to cover the section adequately. Troubleshooting For initial validation of the antibody a positive control test should be performed by conventional IHC as follows: Pretreat the section as needed, including Peroxide Block. Add the primary antibody to the section, incubate for the appropriate time, and wash in PBS. Dilute the Biotin Labeling Reagent ten-fold in PBS and add to the section, incubate for 20 minutes and wash in PBS. Incubate with conjugate, DAB, counterstain and mount as described in the main protocol. The section should show strong tissue-specific staining plus background. This staining pattern should be compared to the Tissue Tackle IHC staining. If background staining is seen with the Tissue Tackle IHC kit, perform the following negative controls to determine the cause of the background staining. Negative Controls If Positive Reaction, then Solution The labeled antibody/mouseblock mixture is omitted. The labeled antibody/mouseblock mixture and the streptavidinenzyme conjugate are omitted. The primary antibody is replaced with an isotype matched non-reactive antibody. The streptavidin-enzyme conjugate is binding non-specifically to the tissue. Endogenous peroxidase activity is present in the tissue. There is non-specific binding of primary antibody (by Fc receptor or other means). Tissue may contain endogenous biotin activity, which can be blocked by treating samples with Avidin (Cat # ) prior to addition of primary antibody. Make sure the tissue sections have been treated with Peroxide Block to inactivate tissue peroxidases. Switch to a different primary antibody or pretreat the sections with an isotype matched non- UHDFWLYHDQWLERG\ JPO Oncogene Research Products Page 6 of 7 Tissue Tackle IHC System

7 Positive Controls If Negative Reaction, then Solution The primary antibody is replaced by an antibody known to react with the test tissue. Fixatives may have reduced access of the antibody to the antigen. Perform target retrieval procedures (either protease digestion or microwave-based methods). Antibody may be too dilute. Increase concentration of the primary antibody by a factor of 10 and/or increase the proportion of Biotin Labeling Reagent by adding 1/5 volume of the Labeling Reagent. The volume of MouseBlock reagent may be reduced to increase signal intensity. References 1. Carson, F.L Histotechnology: A Self-Instructional Text. ASCP Press, Chicago. 2. Elias, J.M Immunohistopathology: A Practical Approach to Diagnosis. ASCP Press, Chicago. 3. Taylor, C.R. and Cote, R.J Immunomicroscopy: A Diagnostic Tool for the Surgical Pathologist. W.B. Saunders Co., Philadelphia. 4. Shi, S.R., et al Cell Vision 2, Reagent Stability All of the reagents included with the Tissue Tackle have been tested for stability. Reagents should not be used beyond the stated expiration date. Power block is a trademark of BioGenex Laboratories. Tween is a tradmark of ICI Americas, Inc CN Biosciences, Inc. Cat# HCS26-60T 22-Aug-01 SMO ONCOGENE RESEARCH PRODUCTS ORDERING INFORMATION Pacific Center Court Research reagents may be ordered on-line, by mail San Diego, CA at the address shown at left, by telephone, Tel: , or fax or from your local Fax: Oncogene Research Products supplier. Terms are net 30 days F.O.B. Web Page: shipping point. For research use only, not for use in diagnostic procedures Oncogene Research Products Page 7 of 7 Tissue Tackle IHC System