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1. important to drain the water from beneath the section by standing the slide vertically on end Dry in an oven at approx 60 C for 30 to 60 minutes E Slides containing smears must contain no large clumps of other accumulations of material that cause lumpiness of the smear surface Any clumps of material will be too thick for adequate microscopic visualization ukhcedata0 1 dept Markey Markey Administration BCP SOP BCP SOP Histology SOP Current Histology SOP Immunohistochemistry BCP H 8 IHC with Vectastain Kit docx University of Kentucky Markey Cancer Center Biospecimen Core Program BCP H 8 113A Combs Building 800 Rose Street Lexington KY 40536 ph 859 257 4717 ph 859 323 2618 fax 859 257 3757 10 10 12 Page 2 of 6 IHC with Vecstain Kit F Smears or cultured cell preparations should be fixed prior to staining Fixed air dried smears or cultures may be stained starting with the first aqueous buffer step of the protocol VI MATERIALS REAGENTS EQUIPMENT and SAFETY PRECAUTIONS A Equipment 1 Tissue Tek containers or coplin jars 2 Forceps 3 Drying oven capable of maintaining 60 C or less 4 Wash bottle 5 Kim Wipes or absorbent wipes 6 Coverslips 7 Slides SuperFrost Plus 8 Timer capable of 3 40 minute intervals 9 Humid chamber B MATERIALS 1 RETRIEVAL BUFFER e g 10X Citrate diluted to appropriate concentration 2 TBST Buffer Solution a For liquid concentrate Mix 250 mill2. Kit Signature Name in Print Date Signature Name in Print Date Signature Name in Print Date Date this version is removed from the manual Date the procedure is retired ukhcedata0 1 dept Markey Markey Administration BCP SOP BCP SOP Histology SOP Current Histology SOP Immunohistochemistry BCP H 8 IHC with Vectastain Kit docx
3. OXYLIN Immerse slides in a bath of hematoxylin Incubate for 2 5 minutes depending on the strength of hematoxylin used Rinse slides in a bath of distilled or deionized water for 2 5 minutes Ensure all residual hematoxylin has been cleared Dip slides 10 times into a bath of 37mmol L ammonia water Rinse slides in a bath of distilled or deionized water for 2 5 minutes Note Depending on the incubation length and potency of the hematoxylin used counterstaining will result in a pale to dark to dark blue coloration of the cell nuclei Excessive or incomplete counterstaining may compromise proper interpretation of results Dehydration and clearing of slides After staining if slides are to be mounted with a non aqueous permanent mounting media you must remove all water before coverslipping This step should be performed at room temperature 20 25 C a Place slides in 95 alcohol and incubate for 5 1 minute Change bath and repeat once b Tap off excess liquid and place slides in 100 alcohol for 3 1 minute Change bath and repeat once c Tap off excess liquid and place slides in xylene or xylene substitute for 3 1 minute Change bath and repeat to 2 times Mounting ukhcedata0 1 dept Markey Markey Administration BCP SOP BCP SOP Histology SOP Current Histology SOP Immunohistochemistry BCP H 8 IHC with Vectastain Kit docx University of Kentucky Markey Cancer Center Biospecimen Core Program BCP H 8 113A Combs Buildi
4. University of Kentucky Markey Cancer Center Biospecimen Core Program BCP H 8 113A Combs Building 800 Rose Street Lexington KY 40536 ph 859 257 4717 ph 859 323 2618 fax 859 257 3757 10 10 12 Page 1 of 6 IHC with Vecstain Kit SUBJECT IHC WITH VECTASTAIN KIT I PRINCIPLE Purpose The ABC system is widely accepted as one of the most sensitive economical and reliable immunoperoxidase systems available The ABC system is based on the extraordinarily high affinity of the glycoprotein avidin for the small molecular weight vitamin biotin Because this affinity is over one million times higher than that of antibody for most antigens the binding of avidin to biotin unlike antibody antigen interactions is essentially irreversible In addition to this high affinity the Biotin Avidin System can be effectively exploited because avidin has four binding sites for biotin and most proteins including antibodies and enzymes can be conjugated with several molecules of biotin These aspects provide the potential for macromolecular complexes to be formed between avidin and biotinylated enzymes An immunoperoxidase procedure based on these properties was devised for localizing a variety of histologically significant antigens and other markers This technique employs unlabeled primary antibody followed by biotinylated secondary antibody and then a pre formed Avidin and Biotinylated horseradish peroxidase macromolecular Complex This system requir
5. ach set of test conditions should be included in each staining run If the positive control tissue fails to demonstrate positive staining results with the test specimen should be considered invalid ukhcedata0 1 dept Markey Markey Administration BCP SOP BCP SOP Histology SOP Current Histology SOP Immunohistochemistry BCP H 8 IHC with Vectastain Kit docx University of Kentucky Markey Cancer Center Biospecimen Core Program BCP H 8 113A Combs Building 800 Rose Street Lexington KY 40536 ph 859 257 4717 ph 859 323 2618 fax 859 257 3757 10 10 12 Page 3 of 6 IHC with Vecstain Kit Negative Control Tissue Use a negative control reagent in place of the primary antibody with a section of each experimental specimen to evaluate nonspecific staining and allow better interpretation of specific staining at the antigen site Refer to the product insert of each primary antibody for specific recommendations The incubation period for the negative control reagent should be the same as the primary antibody SAFETY PRECAUTIONS X PROCEDURE 1 Cut sections to 3 4um thickness Place the sections on positively charged slides or silanized slides DO NOT use additives in the floatation bath Heat slides in oven at 58 to 60 C for 30 to 60 minutes 2 Deparaffinization and Rehydration Prior to staining tissue slides must be deparaffinized to remove embedding medium and rehydrated Avoid incomplete removal of paraffin Residual embed
6. ding medium will result in non specific staining This step should be performed at room temperature 20 25 C a Place slides in a xylene or xylene substitute bath and incubate for 5 1 minute Change bath and repeat to 2 times b Tap off excess liquid and place slides in 100 alcohol for 3 1 minute Change bath and repeat 1 to 2 times c Tap off excess liquid and place slides in 95 alcohol for 3 1 minute Change bath and repeat 1 to 2 times d Tap off excess liquid and place slides in distilled or deionized water for a minimum of 30 seconds Note Xylene and alcohol solutions should be changed after 40 slides 3 If necessary perform antigen retrieval techniques and or enzyme digestion step a Place the enzyme digestion reagent on the tissue for designated time Proteinase K generally 5 min OR a Place slides in a plastic container with Target Retrieval Solution and heat in Decloaking chamber See BCP H 6 Steam Retrieval b Remove the plastic container from the chamber and let stand at room temperature for at least 20 minutes c Place slides in Tris Buffer with Tween for 5 minutes 4 Peroxidase Block Tap off excess buffer Using a lintless tissue such as a Kim wipe carefully wipe around the specimen to remove any remaining liquid and to keep reagents within the prescribed area Apply enough reagent to cover specimens Incubate 5 1 minutes Rinse gently with distilled or deionized water or Wash Buffer from a wa
7. es more time than the standard DAKO Envision system which utilizes a polymer conjugated to secondary antibody and HRP but it does allow researchers to use primary antibodies raised in hosts other than mouse or rabbit Since many research grade antibodies are raised in goat or rat or various other species the flexibility in choice of secondary antibody offsets the additional time required for staining ll ROLE A Research Analyst V SPECIMENS Samples A 10 neutral buffered formalin is the most common fixative Tissues should be fixed for 6 to 12 hours However it should be understood that all fixation procedures result in some reduction of antigen recognition Formalin fixation should be kept to a minimum and the laboratory should test each primary antibody used in a fixation test protocol to verify that the fixation of the specimen is not creating negative specimens from positive ones B The specimen will consist of formalin fixed paraffin embedded FFPE tissues fixed smears or cultured cells adhered to glass slides C Specimens FFPE tissues should be cut into 3 4um thickness unless designated by a specific primary antibody and mounted on positively charged slides D Paraffin sections should be mounted from a preheated floatation bath containing distilled or deionized water bath temperature per institution guidelines The water bath should contain no additives such as gelatin stay on polylysine etc It is
8. iliters buffer with 2 25 Liters of distilled or deionized water Stored at room temperature buffer is good for approximately 2 weeks If buffer becomes cloudy discard immediately 3 Proteinase K Commercially purchased from DakoCytomation S3004 or RTU 3020 with expiration date on the container Store at 2 8 C 4 Hydrogen Peroxide 0 03 hydrogen in methanol 5 Primary Antibody 6 Negative Control Serum rabbit or mouse as appropriate store at 2 8 C Expiration date is on container 7 Biotinylated secondary antibody targeted to appropriate specie Varies based on host in which primary antibody was raised 8 ABC from Vectastain kit Hematoxylin 10 Substrate Chromogen DAB 3 3 diaminobenzidine mix one drop 20uL Liquid DAB Chromogen to 1mL Buffered Substrate Prepared Substrate Chromogen solution is stable for approximately 5 days when stored at 2 8 C This solution should be mixed thoroughly prior to use Any precipitate developing in the solution does not affect staining quality 11 Xylene or Xylene Substitutes 12 Reagent Alcohol 13 Mounting Media Vil QUALITY CONTROL Positive Control Tissue Controls should be fresh surgical specimens autopsy tissue or cell pellets fixed processed and embedded as soon as possible in the same manner as the experimental samples Positive control tissues are indicative of correctly prepared tissue and proper staining techniques One positive control tissue for e
9. in TBST or dilution buffer to cover specimen approximately 200uL At this time the ABC should be prepared see next step Incubate 30 1 minute Rinse gently with wash buffer from a wash bottle do not focus flow directly on tissue and place slides in a fresh wash buffer bath ABC Avidin and biotin require at least 30 minutes at room temperature to complex Be sure to prepare the mixture by the time secondary antibody is applied to slides Add exactly two 2 drops of REAGENT A to 5 ml of buffer mix well then add exactly two 2 drops of REAGENT B to the same bottle mix immediately and allow to stand for about 30 minutes before use Apply enough to cover tissue and incubate at room temperature for 30 minutes Rinse gently with wash buffer from a wash bottle do not focus flow directly on tissue and place slides in a fresh wash buffer bath Substrate Chromogen Solution DAB Tap off excess buffer Using a lintless tissue such as a Kim wipe carefully wipe around the specimen to remove any remaining liquid and to keep reagents within the prescribed area Using a pipette apply enough Substrate Chromogen solution to cover specimen approximately 200uL Incubate for 5 10 minutes or until adequate signal develops Rinse gently with distilled or deionized water only do not focus flow directly on tissue Collect substrate chromogen solution waste in a hazardous materials container for proper disposal Counterstain Directions are for HEMAT
10. ng 800 Rose Street Lexington KY 40536 ph 859 257 4717 ph 859 323 2618 fax 859 257 3757 10 10 12 Page 5 of 6 IHC with Vecstain Kit Non aqueous permanent mounting media are recommended when using DAB as the substrate chromogen Otherwise aqueous mounting media are also acceptable NOTE Slides may be read when convenient However some fading may occur if slides are coverslipped with an aqueous mounting medium and exposed to strong light over a period of one week To minimize fading store slides in the dark at room temperature 20 25 C XIII REFERENCES Vector Laboratories Vectastain Universal Elite ABC Kit User Manual http vectorlabs com data protocols PK 6200 pdf XV HISTORY BLOCK Replaces XVI APPROVAL BLOCK Written by Dana Napier Date 10 10 2012 o y O O Revised by Date Approved by Date _ o oOZ OoOO o Scientific Director Approved by Date Medical Director Annual Review Signed By OOo e O e O Signature sae Signature sae YP Signature ukhedata0 1 dept Markey Markey Administration BCP SOP BCP SOP Histology SOP Current Histology SOP Immunohistochemistry BCP H 8 IHC with Vectastain Kit docx University of Kentucky Markey Cancer Center Biospecimen Core Program BCP H 8 113A Combs Building 800 Rose Street Lexington KY 40536 ph 859 257 4717 ph 859 323 2618 fax 859 257 3757 10 10 12 Page 6 of 6 IHC with Vecstain
11. sh bottle and place in a fresh bath of wash buffer 5 Primary Antibody or Negative Control Reagent Tap off excess buffer Using a lintless tissue such as a Kim wipe carefully wipe around the specimen to remove any remaining liquid and to keep reagents within the prescribed area Apply enough Primary or Negative Control reagent to cover specimen approximately 200uL Incubate for appropriate time at indicated ukhcedata0 1 dept Markey Markey Administration BCP SOP BCP SOP Histology SOP Current Histology SOP Immunohistochemistry BCP H 8 IHC with Vectastain Kit docx University of Kentucky Markey Cancer Center Biospecimen Core Program BCP H 8 113A Combs Building 800 Rose Street Lexington KY 40536 ph 859 257 4717 ph 859 323 2618 fax 859 257 3757 10 10 12 Page 4 of 6 IHC with Vecstain Kit 11 temperature For most antibodies this is 30 minutes to 1 hour at room temperature though other primary antibodies have enhanced staining when incubated overnight at 4 C Refer to the SOP for the particular antibody being used Rinse gently with wash buffer from a wash bottle do not focus flow directly on tissue and place slides in a fresh wash buffer bath Secondary antibody Tap off excess buffer Using a lintless tissue such as a Kim wipe carefully wipe around the specimen to remove any remaining liquid and to keep reagents within the prescribed area Apply enough biotinylated secondary antibody appropriately diluted
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