Cryosection the frozen liver tissue at 5µm thickness and place it on charged slide

Air dry the sections for 3 minutes 3h 0m 0s

Fix the tissue sections in cold acetone for 15 minutes 0h 15m 0s . Alternatively, fix with 1% paraformaldehyde at 4C for 15min followed by ice-cold methanol at -20C for 5min.

Air dry the sections at room temperature for 2 minutes 0h 2m 0s

Incubate the slides in 0.3% hydrogen peroxide in PBS for 5 minutes 0h 5m 0sto block peroxidase activity

Wash slides with PBS 3 times, for 5 minutes each 0h 5m 0s

Block the tissue sections in 10% normal goat serum or 5%Horse serum with 0.1%BSA for 20 minutes 0h 20m 0s

Incubate the tissue sections with primary antibody diluted in DAKO antibody diluent at room temperature for 1.5-2 hours 1h 30m 0s 2h 0m 0s

Wash the slides with PBS 3 times, for 5 minutes each 0h 5m 0s

Incubate the tissue sections with biotinylated secondary antibody diluted in PBS at room temperature for 30-45 minutes 0h 30m 0s 0h 45m 0s

Wash the slides with PBS 3 times, for 5 minutes each 0h 5m 0s

Incubate the tissue sections with ABC (Avidin-Biotin complex) peroxidase solution at room temperature for 30 minutes 0h 30m 0s

Wash the slides with PBS 3 times, 5 minutes each 0h 5m 0s

Incubate the tissue sections with DAB (3,3’-diaminobenzidine) peroxidase substrate solution until desired color intensity is reached and immerse slides in distilled water

Counterstain with hematoxylin and rinse with distilled water

Dehydrate the sections using 95% ethanol followed by 100% ethanol

Clear with xylene and mount coverslip using mounting medium