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Immunohistochemistry (IHC) is a method of detecting the presence of specific proteins in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC is widely used in the diagnosis of abnormal cells and basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue.
This product is based on a novel LSAB (labeled streptavidin-biotin) method. In this new method a single primary antibody is subsequently associated with multiple biotin molecules. Poly-HRP Streptavidin provides multiple active biomolecules on each polymer chain which increase the biotin binding capacity and amplify the peroxidase enzyme signal.
1. RTU normal rabbit serum: 10ml.
2. RTU peroxidase blocking solution: 10ml
3. RTU biotinylated anti-rat secondary antibody: 10ml
4. RTU streptavidin-HRP polymer: 10ml
Store at 2-8°C.
1. Preparation of Slides
A. Cell Lines
Grow cultured cells on sterile glass cover slips or slides overnight at 37 º C
Wash briefly with PBS
Fix as desired. Possible procedures include:
a. 20 minutes with 10% formalin in PBS (keep wet)
b. 10 minutes with ice cold methanol, allow to air dry
c. 10 minutes with ice cold acetone, allow to air dry
Wash in PBS
C. Paraffin Sections
Deparaffinize sections in xylene, 3.5min.
Hydrate with 100% ethanol, 2.2min.
Hydrate with 95% ethanol, 2.2min.
Rinse in distilled water.
Follow procedure for pretreatment as required.
2. Antigen retrieval
Most formalin-fixed tissue requires an antigen retrieval step before immunohistochemical staining can proceed. Heat-mediated and enzymatic antigen retrievals are common methods.
For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer with 0.05% Tween 20, pH 6.0; maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench top for 30 minutes.
For EDTA: Bring slides to a boil in 1 mM EDTA, pH 8.0: follow with 15 minutes at a sub-boiling temperature. No cooling is necessary.
For TE: Bring slides to a boil in 10 mM TE/1 mM EDTA, pH 9.0: then maintain at a sub-boiling temperature for 18 minutes. Cool at room temperature for 30 minutes.
For Pepsin: Digest for 10 minutes at 37°C.
Note: Do not use this pretreatment with frozen sections or cultured cells that are not paraffin-embedded. 3. Staining Procedure
1. Rinse sections in PBS-Triton X-100 (0.025%) for 2.2min
2. Serum Blocking: incubate sections with 3-4 drops of RTU normal rabbit serum for 30 minutes to block non-specific binding of immunoglobulin.
3. Primary Antibody: incubate sections with primary antibody at appropriate dilution in antibody dilution buffer (cat# 6002) for 1-2 hour at room temperature or overnight at 4 °C. Rinse in PBS.
4. Peroxidase Blocking: incubate sections in 3-4 drops of RTU peroxidase blocking solution for 10 minutes at room temperature. Rinse in PBS.
5. Secondary Antibody: incubate sections with 3-4 drops of RTU biotinylated anti-rat secondary antibody for 30 minutes at room temperature.
6. Rinse in PBS for 3.2min.
7. Detection: incubate sections with 3-4 drops of RTU streptavidin-HRP polymer for 30 minutes at room temperature.
8. Rinse in PBS for 3.2min.
9. Chromogen/Substrate: incubate sections with 3 drops of DAB solution for 2-8 minutes. Monitor signal development under a microscope
Note: DAB solution is made by mixture of 1 drop of DAB stock solution with 1ml of DAB buffer which are included in DAB Substrate Kit (Cat# 6003).
10. Rinse in PBS 2.2min.
11. Rinse in distilled water 2.2 min.
12. Counterstain: For using hematoxylin counterstain kit (Cat# 6004), incubate sections with 3 drops of RTU hematoxylin for 1-2 minutes. Rinse in distilled water. Incubate sections with 3 drops of RTU bluing solution for 1 minute.
13. Rinse in distilled water.
14. Dehydrate through75% ethanol for 2 min, 95% ethanol for 2 min, and 100% ethanol for 2.3min. Clear in xylene for 2.5min.
15. Coverslip with mounting medium.
Warning: DAB is a possible carcinogen. Please take necessary precautions.
Note: This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals. Avoid contact with eyes, skin and clothing. Do not ingest. Wear gloves.
Note: All prices in US Dollars