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DakoCytomation LSAB2 System-HRP Code K0672 15 mL Code K0673 15 mL Code K0675 110 mL

Intended use For In Vitro diagnostic use. These instructions apply to the Universal DakoCytomation Labelled Streptavidin-Biotin2 System, Horseradish Peroxidase (LSAB2 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, Bouins, zinc formalin, and neutral buffered formalin may be used. Refer to DakoCytomations General Instructions for Immunohistochemical Staining 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 The LSAB2 System, HRP is based on a modified labeled avidin-biotin (LAB) technique in which a biotinylated secondary antibody forms a complex with peroxidase-conjugated streptavidin molecules.1 In comparison to the ABC method, the LAB/LSAB method has been reported to be four to eight times more sensitive.2 Giorno, et al,2 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. 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 patients clinical history and other diagnostic tests by a qualified individual. The LSAB2 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 510 minute incubation with 3-3' diaminobenzidine (DAB) SubstrateChromogen which results in a brown-colored precipitate at the antigen site. Code K0672 The following materials, sufficient for 150 tissue sections, based on 100 L per section, are included in this kit: Quantity 1x15 mL Description Peroxidase Block 3% hydrogen peroxide in water. 1x15 mL Link Antibody Biotin labelled affinity isolated goat anti-rabbit and goat anti-mouse immunoglobulins in phosphate buffered saline (PBS), containing stabilizing protein and 0.015 mol/L sodium azide. 1x15 mL Streptavidin-HRP Streptavidin conjugated to horseradish peroxidase in PBS containing stabilizing protein and anti-microbial agents. 1x15 mL AEC Substrate Chromogen

Principles of procedure

Reagents provided

AEC in N,N-dimethylformamide (DMF) and acetate buffer, pH 5.0, containing hydrogen peroxide, stabilizers, enhancers and an anti-microbial agent. Keep at 28C.

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Code K0673 The following materials, sufficient for 150 tissue sections, based on 100 L per section, are included in this kit: Quantity 1x15 mL Description Peroxidase Block 3% hydrogen peroxide in water. 1x15 mL Biotinylated Link Biotin labelled affinity isolated goat anti-rabbit and goat anti-mouse immunoglobulins in phosphate buffered saline (PBS), containing stabilizing protein and 0.015 mol/L sodium azide. 1x15 mL Streptavidin-HRP Streptavidin conjugated to horseradish peroxidase in PBS containing stabilizing protein and anti-microbial agents. 1x18 mL Substrate

Imidazole-HCl buffer pH 7.5 containing hydrogen peroxide and an anti-microbial agent. 1x1 mL DAB Chromogen 3,3'-diaminobenzidine in chromogen solution. Accessories 1 Calibrated test tube 1 Plastic Pasteur pipette Code K0675(11) The following materials, sufficient for 1100 tissue sections, based on 100 L per section, are included in this kit: Quantity 1x110 mL Description Biotinylated Link Biotin labelled affinity isolated goat anti-rabbit and goat anti-mouse immunoglobulins in phosphate buffered saline (PBS), containing stabilizing protein and 0.015 mol/L sodium azide. 1x110 mL Streptavidin-HRP Streptavidin conjugated to horseradish peroxidase in PBS containing stabilizing protein and anti-microbial agents. Code K0675(89) The following materials, packaged for use with the Dako Autostainer (code S3400), are included in this kit: Quantity 10x11 mL Description Biotinylated Link Biotin labelled affinity isolated goat anti-rabbit and goat anti-mouse immunoglobulins in phosphate buffered saline (PBS), containing stabilizing protein and 0.015 mol/L sodium azide. 10x11 mL Streptavidin-HRP Streptavidin conjugated to horseradish peroxidase in PBS containing stabilizing protein and anti-microbial agents.

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Materials required, but not supplied

Absorbent wipes Antibodies: N-Series ready-to-use or concentrated antibodies diluted in Antibody Diluent (code S0809 or S3022) Antibody Diluent (code S0809 or S3022) Control tissues, positive and negative Counterstain; aqueous based, such as Lillies Modified Mayers Hematoxylin (code S3309) Coverslips Distilled water Drying oven, capable of maintaining 60C or less. Ethanol, absolute and 95% Light microscope (20x800x) Mounting media, such as Faramount (code S3025) or Glycergel (code C0563) Negative control reagents Slides, poly-L-lysine coated or Silanized Slides (code S3003) Staining jars or baths Timer (capable of 210 minute intervals) Wash bottles Wash buffer solution Xylene, toluene or xylene substitutes For the K0675 LSAB2 System (110 mL), the following reagents are required in addition to the above list: Hydrogen peroxide, 3% solution or Peroxidase Block (code S2001) Substrate-Chromogen solution such as AEC Substrate-Chromogen (code K3464, 110 mL, ready-to-use) or Liquid DAB (code K3466) Optional materials, not supplied Buffers: Wash buffer (code S3006) for automated and manual use Phosphate buffered saline (code S3024) Tris-buffered saline (code S3001 or S1968) Proteolytic Enzymes: Pepsin (code S3002) Proteinase K (code S3004 or S3020) Proteolytic Enzyme, RTU (code S3007) Trypsin (code S2012) Others: Positive Control Slides (available from DakoCytomation) Target retrieval solution (code S1699 or S1700) Target retrieval solution, High pH (code S3307 or S3308) Humidity Chamber Ammonium hydroxide, 15 mol/L diluted to 0.037 mol/L

Precautions

Product Specific 1. For professional users. 2. This product contains sodium azide (NaN3), 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.3,4 3. 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. 4. 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 users laboratory may produce significant variability in results necessitating regular performance of in-house controls. 5. Do not substitute reagents from other lot numbers or from kits of other manufacturers. 6. Incubation times or temperatures other than those specified may give erroneous results; any such changes must be validated by the user. 7. 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.

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General 1. As with any product derived from biological sources, proper handling procedures should be used. 2. Minimize microbial contamination of reagents or incorrect results may occur. 3. Avoid splashing of reagents or generation of aerosols. 4. 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. 5. Safety Data Sheet available for professional users on request. Risk and Safety statements K0672 AEC Substrate Chromogen Ready to Use R61 May cause harm to the unborn child. S53 Avoid exposure obtain special instructions before use. S45 In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible). Restricted to professional users. K0673 DAB Chromogen R40 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. S36/37 Wear suitable protective clothing and gloves. Storage Reagents of the LSAB2 System, HRP are to be stored at 28C. Do not freeze. Do not use after the expiration date printed on reagent vials and kit label. If reagents are stored under any conditions other than those specified, they must be validated by the user.5 The AEC and DAB Substrate-Chromogen solutions are unstable at temperatures greater than 8C. Use and store the AEC and DAB Substrate-Chromogen solutions at the recommended 28C temperature range. These solutions may be used immediately after removal from the refrigerator. After use, return to 28C 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 DakoCytomation Technical Support. Reagent preparation It is convenient to prepare the following reagents prior to staining. Wash Buffer Solution TBST, 0.05 mol/L Tris Buffered Saline with Tween (code S3006) is the recommended wash buffer for automated and manual IHC detection. TBS, 0.05 mol/L Tris Buffered Saline (code S1968) and PBS, 0.02 mol/L Phosphate Buffered Saline (code S3024) are also suitable wash buffer solutions for manual staining. Wash buffer solutions containing sodium azide are not recommended. Sodium azide will inactivate horseradish peroxidase (HRP) resulting in negative staining. Store unused buffer at 28C. Discard buffer if cloudy in appearance. Distilled water may be used for rinsing the hydrogen peroxide, substrate-chromogen solution, and counterstain. Primary Antibody A wide range of N-Series Ready-to-use Primary Antibodies and Negative Control Reagents are available for use with LSAB2 Systems. Concentrated antibodies are also available from DakoCytomation; however, optimal dilutions must be determined experimentally by the user. Dilutions should be prepared using Antibody Diluent (code S0809) which contains 0.05 mol/L Tris-HCl buffer, pH 7.27.6, 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. Antibody Diluent with Background Reducing Components (code 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. 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, 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 DakoCytomation N-Series ready-to-use antibodies, Universal Negative Control(s) is recommended as a negative control reagent. These controls are optimized for use with either mouse (code N1698) or rabbit (code N1699) N-Series ready-to-use antibodies.

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Substrate-Chromogen Solution The K0672 LSAB2 System contains ready-to-use AEC, which is ready to apply to tissue or cell specimens. The K0673 LSAB2 System contains DAB which is prepared as follows: Add 1 drop (or 20 L) of the DAB Chromogen per mL of Substrate Buffer. Use the provided graduated test tube to measure the amount of Substrate Buffer 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 28C. If precipitate forms, mix well before using. Ready-to-use AEC SubstrateChromogen Solution (code K3464, 110 mL) or Liquid DAB (code K3466) is recommended for use with the LSAB2 System, HRP, 110 mL (code K0675). Please follow the instructions provided with each substratechromogen system for substrate-chromogen preparation. 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 Mayers Hematoxylin. Follow counterstaining of hematoxylin with a thorough rinse in distilled water, then immerse tissue slides into a bath of 0.037 mol/L ammonia water. 0.037 mol/L ammonia water is prepared by mixing 2.5 mL of 15 mol/L (concentrated) ammonium hydroxide with 1 liter of water. Unused 0.037 mol/L ammonia water may be stored at room temperature (2025C) in a tightly capped bottle for up to 12 months. Consult manufacturers guidelines for alternative counterstaining procedures. Mounting Media Faramount Aqueous Mounting Medium, ready-to-use (code S3025) or Glycergel Mounting Medium (code C0563) is recommended for aqueous mounting. Liquefy Glycergel by warming to approximately 40 5C prior to use. Specimen preparation Refer to the General Instructions for Immunohistochemical Staining and/or the Antibody Specification Sheet. 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. Staining procedure 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. 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 (2025C) 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 28C for storage after use. Storage at temperatures above 8C 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 (2025C) without affecting staining performance. The sensitivity of the LSAB2 System, HRP can be further increased by lengthening the incubation times of Steps 2, 3 and 4 to 30 5 minutes each. Staining Protocol STEP 1 PEROXIDASE BLOCK 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.

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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. 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 (2025C) 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 28C storage. For DAB preparation, refer to Reagent Preparation Section. 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 510 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 (25) minutes, depending on the strength of hematoxylin used. Rinse gently in a distilled water bath. Dip slides 10 times into a bath of 0.037 mol/L ammonia water (optional). Rinse slides in a bath of distilled or deionized water for two to five (25) minutes. STEP 7 MOUNTING Specimens may be mounted and coverslipped with an aqueous-based mounting medium such as Faramount (code S3025) or Glycergel (code 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 (2025C). Quality control Differences in tissue processing and technical procedures in the users laboratory may produce significant variability in results, necessitating regular performance of in-house controls. Consult the quality control guidelines of the College of American Pathologists (CAP) Certification Program for Immunohistochemistry and references 68 for additional information. Refer to the package insert of each primary antibody used for details regarding sensitivity and immunoreactivity. Refer to the General Instructions for Immunohistochemical Staining for further information on positive and negative controls. Staining interpretation General limitations Refer to the General Instructions for Immunohistochemical Staining for interpretation guidelines. Refer to the General Instructions for Immunohistochemical Staining for general limitations.

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Product specific limitations

DakoCytomation provides LSAB2 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).9-12 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 7.27.6, prior to staining or use Biotin Blocking System (code X0590). Endogenous peroxidase or pseudoperoxidase activity can be found in hemoproteins such as hemoglobin, myoglobin, cytochrome and catalase as well as in eosinophils.13,14 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 Peroxidase Blocking Reagent (code 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.8 Contact DakoCytomation Technical Support 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.15

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Troubleshooting

Problem 1. No staining of any slides.

Probable Cause 1a. Reagents not used in proper order. 1b. Sodium azide in buffer bath. 1c. Substrate-chromogen reagent mixed incorrectly or not working. 2a. Sections retain too much solution after wash bath. 2b. Slides not incubated long enough with antibodies or mixture. 2c. Incompatible counterstain or mounting media that dissolves reaction product. 3a. Specimens contain high endogenous peroxidase activity. 3b. Paraffin incompletely removed. 3c. Slides not properly rinsed. 3d. Faster than normal substrate reaction due to excessive room temperature. 3e. Sections dried during staining procedure. 3f. Nonspecific binding of reagents to tissue section.

Suggested Action 1a. Review application of reagents. 1b. Use fresh, azide-free buffer. 1c. Test prepared substrate-chromogen with a drop of the streptavidin solution. If no color reaction occurs, make a fresh substrate-chromogen solution according to kit protocol. 2a. Gently tap off excess solution before wiping around section. 2b. Review recommended incubation times. 2c. For AEC immunostained slides, use only aqueous-based counterstains and mounting media. 3a. Incubate slides with fresh hydrogen peroxide. 3b. Use fresh xylene or toluene baths. If several slides are stained simultaneously, the second xylene bath should contain fresh xylene. 3c. Use fresh solutions in buffer baths and wash baths. 3d. Use shorter incubation time with substrate-chromogen solution. 3e. Use humidity chamber. Wipe only three to four slides at a time before applying reagent. 3f. Use DakoCytomation antibody diluents or add 1% BSA to the antibody diluent. Alternatively, 0.05 mol/L Tris containing 0.3 mol/L NaCl and 0.1% Tween 20, pH 7.2-7.6 (code S3306) may be used as a wash buffer. The incubation of a separate protein block reagent (code X0909) prior to the application of the primary antibody may also be necessary. 3g. Use higher dilution of the primary antibody. 4a. Use poly-L-lysine slides for most staining. For primary antibodies that require target retrieval techniques, use Silanized Slides. 4b.Try a different lot number of the Silanized Slides. 5a. Do serial dilutions of primary antibody to determine optimum dilution. 5b. Determine the appropriate staining protocol for the antibody, i.e. length of primary antibody incubation, biotinylated link and streptavidin-HRP length of incubation.

2. Weak staining of all slides.

3. Excessive background staining in all slides, including control negative slides.

4. Tissue sections detaching from slides.

3g. Primary antibody too concentrated. 4a. Use of incorrect slides.

5. Excessively strong specific staining.

4b. Slides not properly prepared prior to tissue mounting. 5a. Use of too much antibody. 5b. Incubation for primary antibody, biotinylated link or streptavidin-HRP too long.

Note: If the problem cannot be attributed to any of the above causes, or if the suggested corrective action fails to resolve the problem, please call DakoCytomation Technical Support for further assistance. Additional information on staining techniques and specimen preparation can be found in the Handbook Immunochemical Staining Methods10 (available from DakoCytomation), Atlas of Immunohistology16 and Immunoperoxidase Techniques, A Practical Approach to Tumor Diagnosis17

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References

1. Guesdon JL, et al. The use of avidin-biotin interaction in immunoenzymatic techniques. J Histochem Cytochem 1979; 27:1131 2. Giorno R. A comparison of two immunoperoxidase staining methods based on the avidin-biotin interaction. Diag Immunol 1984; 2:161 3. Center for Disease Control Manual Guide Safety Management, No. CDC-22. Decontamination of laboratory sink drains to remove azide salts. Atlanta, Georgia. April 30, 1976 4. 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 5. Clinical Laboratory Improvement Amendments of 1988: Final rule, 57FR7163. February 18, 1992 6. Elias JM, et al. Special report: Quality control in immunohistochemistry. Amer J Clin Pathol 1989; 92:836 7. National Committee for Clinical Laboratory Standards. Internal quality control testing: Principles and definitions; approved guideline. Villanova, PA. 1991; 4: Order code C24-A 8. Herman GE and Elfont EA. The taming of immunohistochemistry: The new era of quality control. Biotech & Histochem 1991; 66:194 9. Kiernan JA. Histological and histochemical methods: Theory and practice. New York: Pergamon Press 1981:81 10. Naish SJ (ed). Handbook: Immunochemical staining methods. Carpinteria: DakoCytomation 1989 11. 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; 29:1196 12. Banerjee D and Pettit S. Endogenous avidin-binding activity in human lymphoid tissue. J Clin Pathol 1984; 37:223 13. Escribano, LMetal. Endogenous Peroxidase activity in human cutaneous and adenoidal mast cells. J Histochen Cytochem 1987; 66:194 14. Elias JM. Immunohistopathology: A practical approach to diagnosis. Chicago: American Society of Clinical Pathologists Press 1990; 46 15. 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; 73:626 16. Tubbs RR, et al. Atlas of immunohistology. Chicago: ASCP Press 1986 17. Nadji M and Morales AR. Immunoperoxidase techniques, A practical approach to tumor diagnosis. Chicago: ASCP Press 1986

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