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 .
