Immunohistochemistry Protocol for Paraffin-Embedded Sections
Fix freshly dissected tissue (<3mm thick) with 10% formalin or other fixatives for 24-48 h at room temperature.
Rinse the tissue with running tap water for 1 h. Dehydrate the tissue through 70% alcohol, 45 min.
Clear the tissue through a change of xylene, 1 h. (1/2) 
Clear the tissue through a change of xylene, 1 h. (2/2) 
Immerse the tissue in a change of paraffin, 1 h. (1/3) 
Immerse the tissue in a change of paraffin, 1 h. (2/3) 
Immerse the tissue in a change of paraffin, 1 h. (3/3) 
Embed the tissue in a paraffin block.
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.
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.
Deparaffinize slides in a change of xylene, 5 min. (1/2) 
Deparaffinize slides in a change of xylene, 5 min. (2/2) 
Drain off the blocking buffer from the slides.
Transfer slides to 100% alcohol, for a 3-minute change. (1/2) 
Transfer slides to 100% alcohol, for a 3-minute change. (2/2) 
Block endogenous peroxidase activity by incubating sections in 3% H2O2 solution in methanol at room temperature for 10 min to block endogenous peroxidase activity.
Change by rinsing in 300 ml of PBS, for 5 min. (1/2) 
Change by rinsing in 300 ml of PBS, for 5 min. (2/2) 
(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.
Change by rinsing slides in 300 ml PBS for 5 min. (1/2) 
Change by rinsing slides in 300 ml PBS for 5 min. (2/2) 
(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 1h.
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 h. 
Change by washing the slides in 300 ml PBS for 5 min. (1/2) 
Change by washing the slides in 300 ml PBS for 5 min. (2/2) 
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.
Change by washing slides in 300 ml PBS for 5 min. (1/2) 
Change by washing slides in 300 ml PBS for 5 min. (2/2) 
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).
Change by washing slides in 300 ml PBS for 5 min. (1/2) 
Change by washing slides in 300 ml PBS for 5 min. (2/2) 
Apply 100 µl 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 < 5 min until the desired color intensity is reached.
Change by washing slides in 300 ml PBS for 2 min. (1/3) 
Change by washing slides in 300 ml PBS for 2 min. (2/3) 
Change by washing slides in 300 ml PBS for 2 min. (3/3) 
(optional) Counterstain slides by immersing sides in Hematoxylin (e.g. Gill’s Hematoxylin) for 1-2 min.
Rinse the slides in running tap water for > 15 min.
Dehydrate the tissue slides through a change of 95% alcohol, for 5 min. (1/2) 
Dehydrate the tissue slides through a change of 95% alcohol, for 5 min. (2/2) 
Clear the tissue slides in a change of xylene and coverslip using mounting solution (e.g. Permount). (1/3) 
Clear the tissue slides in a change of xylene and coverslip using mounting solution (e.g. Permount). (2/3) 
Clear the tissue slides in a change of xylene and coverslip using mounting solution (e.g. Permount). (3/3)
Observe the color of the antibody staining in the tissue sections under microscopy.
Dehydrate the tissue through 80% alcohol, 45 min.
Dehydrate the tissue through 95% alcohol, 45 min.
Dehydrate the tissue through a change of 100% alcohol, 1 h. (1/3) 
Dehydrate the tissue through a change of 100% alcohol, 1 h. (2/3) 
Dehydrate the tissue through a change of 100% alcohol, 1 h. (3/3) 
Transfer through 95% alcohol for 3 min.
Transfer through 70% alcohol for 3 min.
Transfer through 50% alcohol for 3 min.
Dehydrate the tissue slides through a change of 100% alcohol, for 5 min. (1/2) 
Dehydrate the tissue slides through a change of 100% alcohol, for 5 min. (2/2) 
