Midified Immunohistochemical Protocol for Paraffin Embedded Sections
1. Deparaffinize and rehydrate sections:
3 x 3´ Xylene
3 x 2´ 100% Ethanol
1 x 2´ 95%, 80%, 70% Ethanol (each)
1 x 5´ 1X PBS
2. Antigen retrieval methods:
Sodium Citrate Antigen Retrieval:
a. Place slides in a glass slide holder and fill in the rest of the rack with blank
slides (10 total) to ensure even heating.
b. Place rack in 600 ml of 10 mM Sodium Citrate (pH 6.0, 100 mM stock) in a
glass 2L beaker. Mark a line at the top of the liquid on the beaker.
c. Microwave for 20 minutes total, replacing evaporated water every 10 min.
d. Cool slides for 20 minutes in the beaker.
e. Wash 3 x 5´ in dd H2O, 1 x 5´ in 1X PBS.
Proteinase K Antigen Retrieval:
a. Make a fresh solution of: 25 ul of 20 mg/ml Proteinase K
2.5 ml of 1 M Tris-Cl, pH 8.0
0.5 ml of 0.5 M EDTA, pH 8.0
to 50 mls with dd H2O
b. Incubate slides in solution at 37°C for 5 min (do NOT pre-warm Prot K
solution). A Coplin staining jar works well for this step.
c. Wash 3 x 5´ with 1X PBS.
Urea Antigen Retrieval:
a. Make a fresh solution of 1 M urea
b. Place slides in a glass slide holder and fill in the rest of the rack with blank
slides (10 total) to ensure even heating.
c. Place rack in 600 ml of 1 M urea in a glass 2L beaker. Mark a line at the top
of the liquid on the beaker.
d. Microwave for 10, 20 or 30 minutes total, replacing evaporated water every
5-10 minutes.
e. Cool slides for 30 minutes to 1 hour in the beaker.
f. Wash 3 x 5´ in dd H2O, 1 x 5´ in 1X PBS.
Trypsin antigen retrieval protocol
a. Place slides or specimens into rack or other suitable container. Slides from paraffin-embedded samples should be dewaxed and rehydrated.
b. Incubate the specimens for 10 minutes at room temp in PBS with occasional agitation to thoroughly hydrate the specimen.
c. Pour off the PBS and incubate the specimen in a solution of 0.1% trypsin (tissue culture grade), 0.1% calcium chloride, 20 mM Tris (pH 7.6-8.0) solution for 2-20 minutes at room temperature depending upon the thickness and type of tissue. Optimization will be necessary. Typical time is 5 minutes.
d. Stop the digestion by gently rinsing the specimen under the cold tap for 5 minutes, followed by incubation in TBS or PBS.