TITLE: Immunohistochemistry Protocol for Paraffin-Embedded Sections
AUTHORS: Sam Li

[DESCRIPTION]
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[STEPS]
?. [Prepare formalin-fixed, paraffin-embedded tissue sections (steps 1-8):]
Fix freshly dissected tissue (less than 3 mm thick) with 10% formalin or other fixatives for 24-48 hour at room temperature. Caution: Formalin is a suspected carcinogen. It can cause eye, skin, and respiratory tract irritation. It should be handled in a hood.
?. [Prepare formalin-fixed, paraffin-embedded tissue sections (steps 1-8):]
Rinse the tissue with running tap water for 1 hour.
?. [Prepare formalin-fixed, paraffin-embedded tissue sections (steps 1-8):]
Dehydrate the tissue through 70%, 80%, and 95% alcohol, 45 min each, followed by 3 changes of 100% alcohol, 1 hour each.
?. [Prepare formalin-fixed, paraffin-embedded tissue sections (steps 1-8):]
Clear the tissue through 2 changes of xylene, 1 hour each.
?. [Prepare formalin-fixed, paraffin-embedded tissue sections (steps 1-8):]
Immerse the tissue in 3 changes of paraffin, 1 hour each.
?. [Prepare formalin-fixed, paraffin-embedded tissue sections (steps 1-8):]
Embed the tissue in a paraffin block. The paraffin tissue block can be stored at room temperature for years.
?. [Prepare formalin-fixed, paraffin-embedded tissue sections (steps 1-8):]
Section the paraffin-embedded tissue block at 5-8 µm thickness on a microtome and float in a 40°C water bath containing distilled water.
?. [Prepare formalin-fixed, paraffin-embedded tissue sections (steps 1-8):]
Transfer the sections onto glass slides suitable for immunohistochemistry (e.g. Superfrost Plus). Allow the slides to dry overnight and store slides at room temperature until ready for use.
?. [Immunostain formalin-fixed, paraffin-embedded tissue sections (steps 9-29):]
Deparaffinize slides in 2 changes of xylene, 5 min each.
?. [Immunostain formalin-fixed, paraffin-embedded tissue sections (steps 9-29):]
Transfer slides to 100% alcohol, for 2 changes, 3 min each, and then transfer once through 95%, 70%, and 50% alcohols respectively for 3 min each.
?. [Immunostain formalin-fixed, paraffin-embedded tissue sections (steps 9-29):]
Block endogenous peroxidase activity by incubating sections in 3% H2O2 solution in methanol at room temperature for 10 min to block endogenous peroxidase activity.
?. [Immunostain formalin-fixed, paraffin-embedded tissue sections (steps 9-29):]
Rinse in 300 ml of PBS for 2 changes, 5 min each.
?. [Immunostain formalin-fixed, paraffin-embedded tissue sections (steps 9-29):]
Optional: Perform antigen retrieval to unmask the antigenic epitope. The most commonly used antigen retrieval is a citrate buffer method. Arrange the slides in a staining container. Pour 300 ml of 10 mM citrate buffer, pH 6.0 into the staining container and incubate it at 95-100°C for 10 min (optimal incubation time should be determined by user). Remove the staining container to room temperature and allow the slides to cool for 20 min.
?. [Immunostain formalin-fixed, paraffin-embedded tissue sections (steps 9-29):]
Rinse slides in 300 ml PBS for 2 changes, 5 min each.
?. [Immunostain formalin-fixed, paraffin-embedded tissue sections (steps 9-29):]
Optional: Add 100 µl blocking buffer (e.g. 10% fetal bovine serum in PBS) onto the sections of the slides and incubate in a humidified chamber at room temperature for 1 hour.
?. [Immunostain formalin-fixed, paraffin-embedded tissue sections (steps 9-29):]
Drain off the blocking buffer from the slides.
?. [Immunostain formalin-fixed, paraffin-embedded tissue sections (steps 9-29):]
Apply 100 µl appropriately diluted primary antibody (in antibody dilution buffer, e.g. 0.5% bovine serum albumin in PBS) to the sections on the slides and incubate in a humidified chamber at room temperature for 1 hour.
?. [Immunostain formalin-fixed, paraffin-embedded tissue sections (steps 9-29):]
Wash the slides in 300 ml PBS for 2 changes 5 min each.
?. [Immunostain formalin-fixed, paraffin-embedded tissue sections (steps 9-29):]
Apply 100 µl appropriately diluted biotinylated secondary antibody (using the antibody dilution buffer) to the sections on the slides and incubate in a humidified chamber at room temperature for 30 min.
?. [Immunostain formalin-fixed, paraffin-embedded tissue sections (steps 9-29):]
Wash slides in 300ml PBS for 2 changes, 5 min each.
?. [Immunostain formalin-fixed, paraffin-embedded tissue sections (steps 9-29):]
Apply 100 µl appropriately diluted Sav-HRP conjugates (using the antibody dilution buffer) to the sections on the slides and incubate in a humidified chamber at room temperature for 30 min (keep protected from light).
?. [Immunostain formalin-fixed, paraffin-embedded tissue sections (steps 9-29):]
Wash slides in 300ml PBS for 2 changes, 5 min each.
?. [Immunostain formalin-fixed, paraffin-embedded tissue sections (steps 9-29):]
Apply 100µl DAB substrate solution (freshly made just before use: 0.05% DAB - 0.015% H2O2 in PBS) to the sections on the slides to reveal the color of antibody staining. Allow the color development for less than 5 min until the desired color intensity is reached. Caution: DAB is a suspected carcinogen. Handle with care. Wear gloves, lab coat and eye protection.
?. [Immunostain formalin-fixed, paraffin-embedded tissue sections (steps 9-29):]
Wash slides in 300 ml PBS for 3 changes 2 min each.
?. [Immunostain formalin-fixed, paraffin-embedded tissue sections (steps 9-29):]
Optional: Counterstain slides by immersing sides in Hematoxylin (e.g. Gill's Hematoxylin) for 1-2 min.
?. [Immunostain formalin-fixed, paraffin-embedded tissue sections (steps 9-29):]
Rinse the slides in running tap water for more than 15 min.
?. [Immunostain formalin-fixed, paraffin-embedded tissue sections (steps 9-29):]
Dehydrate the tissue slides through 4 changes of alcohol (95%, 95%, 100%, and 100%), 5 min each.
?. [Immunostain formalin-fixed, paraffin-embedded tissue sections (steps 9-29):]
Clear the tissue slides in 3 changes of xylene and coverslip using mounting solution (e.g. Permount). The mounted slides can be stored at room temperature permanently.
?. [Immunostain formalin-fixed, paraffin-embedded tissue sections (steps 9-29):]
Observe the color of the antibody staining in the tissue sections under microscopy.