Immunohistochemistry Protocol for Frozen Sections
Fix the tissue sections with a suitable fixative.
One of the commonly used fixation methods for frozen tissue sections is to immerse the slides in pre-cooled acetone (-20°C) for 10 min.
Place a freshly dissected tissue block (<5 mm thick) on to a pre-labeled tissue base mold.
Cover the entire tissue block with cryo-embedding media (e.g. OCT).
Slowly place the base mold containing the tissue block into liquid nitrogen till the entiretissue block is submerged into liquid nitrogen to ensure tissue is frozen completely.
Store the frozen tissue block at -80°C until ready for sectioning.
Transfer the frozen tissue block to a cryotome cryostat (e.g. -20°C) prior to sectioning and allow the temperature of the frozen tissue block to equilibrate to the temperature of the cryotome cryostat.
Section the frozen tissue block into a desired thickness (typically 5-10 µm) using the cryotome.
Place the tissue sections onto glass slides suitable for immunohistochemistry (e.g. Superfrost).
Dry the tissue sections overnight at room temperature.
Sections can be stored in a sealed slide box at -80°C for later use.
Pour off the fixative and allow acetone to evaporate from the tissue sections for ≥ 20 min at room temperature.
Rinse the slides in 300 ml of 10 mM phosphate buffered saline (PBS) at a neutral pH for 5 min (1/2).
Rinse the slides in 300 ml of 10 mM phosphate buffered saline (PBS) at a neutral pH for 5 min (2/2).
Incubate the slides in 0.3% H2O2 solution in PBS at room temperature for 10 min to block endogenous peroxidase activity.
Rinse the slides in 300 ml PBS for 5 min (1/2).
Rinse the 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.
Drain off the blocking buffer from the slides.
Apply 100 µl an 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 for 1 h at room temperature or overnight at 4°C.
Rinse the slides in 300 ml PBS for 5 min (1/2).
Rinse the slides in 300 ml PBS for 5 min (2/2).
Apply 100 µl an appropriately diluted biotinylated secondary antibody (using the antibodydilution buffer) to the sections on the slides and incubate in a humidified chamberat room temperature for 30 min.
Rinse the slides in 300 ml PBS for 5 min (1/2).
Rinse the slides in 300 ml PBS for 5 min (2/2).
Rinse the slides in 300 ml PBS for 5 min (1/2).
Rinse the 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 the antibody staining.
Allow the color development for ≤ 5 min until the desired color intensity is reached.
Wash slides in 300 ml PBS for 5 min (1/2).
Wash slides in 300 ml PBS for 5 min (2/2).
(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 with  95% alcohol change (1/4).
Dehydrate the tissue slides with 95% alcohol change (2/4).
Dehydrate the tissue slides with 100% alcohol change (3/4).
Dehydrate the tissue slides with 100% alcohol change (4/4).
Add 100 µl pre-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).
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.
