INSTRUCTIONS FOR IMMUNOHISTOCHEMICAL STAINING INTRODUCTION

 

Reagents supplied (for 100 sections)
component size  storage
Blocking Serum 20ml Store at 4°C for 1year
Biotinylated,Affinity-purified Anti-Rabbit IgG 220ul Store at 4°C for 1year
Reagent A (Streptavidin) 210ul Store at 4°C for 1year
Reagent B (Biotinylated Horseradish Peroxidase) 210ul Store at 4°C for 1year
  • NOTE: reagents not supplied (Primary Antibody, Buffer, Hydrogen Peroxide, Oxidizable Peroxidase Substrate)
PREPARATION OF FineTest WORKING SOLUTIONS
  • A number of different buffers can be used in the FineTest SABC system. One of the most common is 10 mM sodium phosphate, pH 7.4, 0.9% saIine (PBS). The SABC working solutions are prepared as follows:
  • Blocking Serum working solution: add 2ml of stock Blocking Serum to 10 ml of PBS. The preferred serum for blocking is prepared from the same species in which the biotinylated secondary antibody is made.
  • Biotinylated Antibody working solution: add 100ul Biotinylated Antibody to 10 ml of Blocking Serum working solution (PBS contain Blocking Serum).
  • SABC Reagent working solution: Mix 100ul of Reagent A and 100ul Reagent B in a EP tube, immediately. Then place it on 37°C for 30 Minutes, add the mixture into 10 ml of Blocking Serum working solution(PBS contain Blocking Serum)for using. It is best to use SABC Reagent working solution within 12 hours.
  • The configuration table
  Blocking SerumWorking solution Biotinylated AntibodyWorking solution SABC ReagentWorking solution
1 section 200ul-400ul 200ul 200ul
10 sections 2ml-4ml 2ml 2ml
100 sections 20ml-40ml 20ml 20ml
STAINING PROCEDURE FOR PARAFFIN SECTIONS

Deparaffinize and hydrate tissue sections through xylenes or other clearing agents and graded alcohol series.

Rinse for 5 minutes in tap water.

If quenching of endogenous peroxidase activity is required, incubate the sections for 30 minutes in 0.3% H2O2 in methanol or water. Incubation times may be shortened by using higher concentrations of H2O2. If endogenous peroxidase activity does not present a problem, step 3 may be deleted.

Wash in buffer for 5 minutes.

Incubate sections for 60 minutes with diluted Blocking Serum working solution from the species in which the secondary antibody is made. (In cases where non-specific staining is not a problem, Steps 5 and 6 may be deleted).

Blot excess Blocking Serum working solution from sections.

Incubate sections for 60 minutes with primary antiserum diluted in buffer. (If background staining occurs, dilutions of the primary and secondary antibodies may be made in buffer containing 1-2% of the appropriate blocking serum.)

Wash slides for 5 minutes in buffer.

Incubate sections for 60 minutes with diluted biotinylated secondary antibody solution.

Wash slides for 5 minutes in buffer.

Incubate sections for 30 minutes with FineTest SABC Reagent working solution

Wash slides for 5 minutes in buffer.

Incubate sections in peroxidase substrate solution until desired stain intensity develops. (See Note 1)

Rinse sections in tap water. 15. Counterstain, clear and mount.

NOTES

Solutions containing sodium azide or other inhibitors of peroxidase activity should not be used in diluting the peroxidase substrate or the SABC Reagent. Do not add normal serum, non-fat dried milk, culture media, or other potential sources of biotin to the SABC reagent. This may result in reduced sensitivity.

Development times may differ depending upon the level of antigen, the intensity of the stain that is required, or the substrate used. DAB generally should be developed for 2-10 minutes; AEC for 10-30 minutes; TMB for 5-20 minutes. Some counterstains may not be compatible with certain peroxidase substrates because of solubility of the reaction products or lack of color contrast. A counterstain compatibility chart is available upon request.

If the reagents are to be diluted beyond their recommended concentrations, first prepare the diluted biotinylated antibody and SABC reagent as described in the instructions. Subsequent dilutions should be made in a buffer containing 0.1% immunohistochemical grade bovine serum albumin. Only immunohistochemical grade BSA should be used, as other preparations can containundesired impurities. Dilution of these reagents may require longer incubation times and/or higher incubation temperatures to achieve maximum sensitivities.

The section should be well prepared. Fixation (generally, in buffered formalin not exceeding 4 percent formaldehyde) should be sufficient to maintain the integrity of the section throughout the staining procedure but not so harsh as to destroy the antigen under study. During the staining procedure, do not allow the section to dry out. Use a humidified chamber for incubations.

To avoid adsorption of the antibody to the plastic or glass container in which the final dilution is made, the primary antibody may be diluted in buffers containing 0.1% immunohistochemical grade bovine serum albumin or dilute Blocking Serum.

Incubation times may be shortened. In cases where the antigen concentration in the section is high, suggested incubation times with primary antibody, biotinylated secondary antibody, and SABC Reagent may be reduced. Incubation times as short as five minutes have been reported to be sufficient in some cases when incubation temperatures are raised to 37 °C. If the antigen concentration is low, steps 7 and 9 may be lengthened to achieve maximal staining

SABC Kit (Rabbit specific)

SKU: MD-IHC0002
Size
Application
Manufacture
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