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Lab Standard Operating Procedure (SOP) for Immunohistochemical Staining
for nuclear factor kappa B (NFkB)
Materials
Literature
1.
Cut paraffin sections @ 4 microns and place on positively-charged
slides (Surgipath X-tra Slides® 00210)
2. Microwave slides briefly 1-2 min. on high power until paraffin
is just melted or bake in 60 degree C oven for 1 hour.
 3.
Deparaffinize , run slides to water and perform steamer (Black and
Decker HS2000 vegetable steamer) retrieval @ 90-100 C in Dako Target
Retrieval solution (S1699) for 30 min. with 20 min. room temperature
(RT) cooling time. Distilled water rinse several times.
4.
Quench endogenous peroxidase with fresh 3% H202 for 5 min.
5.
Rinse with distilled water several times and place in Tris with
Tween buffer (Dako S3306)
6.
Block tissue with A/B block (Vector) for 15 min. each with a Tris
withTween buffer rinse between and after.
7.
Wipe around tissue and block with protein block (Dako X0909 ) for
10 min.
8.
Drip protein block off slide and apply primary antibody for overnight
incubation. Primary antibody = mouse monoclonal IgG1 recombinant
amino-terminal 1-268 human nuclear factor kappa B (NFkB) p65
(source: Santa Cruz, SC-8008), 1:800 dilution. The antibody can
be used on formalin-fixed paraffin-embedded tissue using pig ovary
as positive control.
9.
Next day place slides in Tris with Tween buffer and rinse once.
10.
Wipe around tissue and apply secondary antibody for 30 min. incubation
at RT. (Dako, LSAB+ Kit, K0690;)
11.
Tris with Tween buffer rinse.
12.
Apply label (Streptavadin, Dako , LSAB+ Kit K0690) for 30 min. at
RT
13.
Rinse with distilled water several times.
14.
Wipe around slides and apply diaminobenzidine (DAB Dako, K3466)
for 5-10 min., monitor slides and place in water when desired intensity
has been reached.
15.
Rinse several times with distilled water.
16.
Apply DAB enhancer (Dako, S1961) for 5 min. at RT.
17.
Rinse several times with distilled water.
18.
Give a 1 min. hematoxylin counterstain followed by 10 seconds in
1% acid alcohol and 1 min. in ammonia water to blue.
19.
Run through graded alcohols to xylene and coverslip with permanent
mounting media.
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