immunohistochemical staining (ihc) - genetex · protocol: immunohistochemical staining (lhc)...
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Protocol: Immunohistochemical staining (IHC)
MATERIAL
Tissue sections
Primary antibody
Conjugated secondary antibody
ABC reagents
Xylene
100% ethanol
95% ethanol
3% hydrogen peroxide
B U F FER 5
PBS
Antigen retrieval buffer: 10 mM sodium citrate buffer pH 6.0
1 mM EDTA pH 8.0
Pepsin
Blocking buffer: 5-10% normal animal serum
METHOD1:IHC-P (for paraffin embedded tissue)
I. Oeparaffinize/hydrate sections
1. Xylene: twice, each for 5 min
2. 100% ethanol: twice, each for 3 min
3. 95% ethanol : 3 min
4. 70 % ethanol: 3 min
5. 50 % ethanol : 3 min
6. Wash slides in deionized water for 3 min
II. Antigen Unmasking
1. Bring slides to a boil in antigen retrieval solution for 15 min in a microwave. Cool slides on bench top for 20 min, or
2. Pepsin digest for 10 min at 3TC.
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Protocol: Immunohistochemical staining (lHC)
III. Staining
1. Wash sections in dH20 twice, each for 3 min.
2. Incubate sections in 3% hydrogen peroxide for 10 min.
3. Wash section in PBS twice, each for 3 min.
4. Block each section with blocking buffer for 20 min at RT.
5. Remove blocking buffer and add primary antibody at proper dilution and incubate for one hour at RT or overnight at 4 "C.
6. Remove antibody solution and wash sections in PBS three times, each for 3 min.
7. Add secondary antibody, and incubate for 30 min at RT.
8. (Prepare ABC reagent according to the manufacturer's instructions and mix solution for 30 min at RT).
9. Remove secondary antibody solution and wash sections in PBS three times, each for 3 min.
10. Add ABC reagent to each section and incubate for 30 min at RT.
11. Remove ABC reagent and wash sections in PBS three times ,each for 3 min.
12. Add DAB to each section and monitor staining closely.
13. As soon as the sections develop, immerse slides in dH20.
14. Counter-stain sections in hematoxylin.
15. Wash sections in dH20 twice, each for 3 min.
16. Dehydrate sections:
Incubate sections in 95% ethanol for twice, each for 3 min.
Incubate sections in 100% ethanol twic,e each for 3 min.
Incubate sections in xylene twice, each for 3 min.
17. Mount sections with coverslip.
Method 2: IHC-F (for frozen tissue)
1. Fix section by immersing in acetone jar for 1-2 min at RT, let air dry.
2. Wash sections in dH20 for twice, each for 3 min.
3. Incubate sections in 0.3% hydrogen peroxide for 10 min.
4. Wash section in PBS for twice each for 3 min.
5. Block each section with blocking solution for 20 min at RT.
6. Remove blocking solution and add primary antibody at proper dilution and incubate for one hour at RT or
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Protocol: Immunohistochemical staining (lHC)
overnight at 4'C.
7. Remove antibody solution and wash sections in PBS three times, each for 3 min.
8. Add secondary antibody, and incubate for 30 min at RT.
9. (Prepare ABC reagent according to the manufacturer's instructions and mix solution for 30 min at RT).
10. Remove secondary antibody solution and wash sections in PBS three times, each for 3 min.
11. Add ABC reagent to each section and incubate for 30 min at RT.
12. Remove ABC reagent and wash sections in PBS three times, each for 3 min.
13. Add DAB to each section and monitor staining closely.
14. As soon as the sections develop, immerse slides in dH20.
15. Counter-stain sections in hematoxylin
16. Wash sections in dH20 twice, each for 3 min.
17. Dehyd rate sections:
Incubate sections in 95% ethanol twice, each for 3 min.
Incubate sections in 100% ethanol twice, each for 3 min.
Incubate sections in xylene twice, each for 3 min.
18. Mount sections with coverslip.
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