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 )
