Post mortem human substantial nigra TH staining
Xiqun Chen, Waijiao Kai
Abstract
This protocol is standardized for postmortem (frozen) SN tissue from pathologically diagnosed PD patients and control individuals for Immunofluorescence staining.
Steps
Deparaffinization
Wash slides in 2 changes of 300mL ml of xylene – 0h 6m 0s each at room temperature.
Rehydration
Wash slides in 300mL of 100% alcohol –0h 9m 0s at Room temperature.
Wash slides in300mL of 95% alcohol – 0h 6m 0s at Room temperature.
Wash slides in 300mL of 80% alcohol – 0h 3m 0s at Room temperature.
Rinse slides in gentle running distilled water – 0h 5m 0s atRoom temperature.
Antigen retrieval
Microwave (high power) slides in 200mL 0.01Molarity (M) sodium citrate buffer, pH 6.0 at 99-100°C for 0h 5m 0s, three rounds.
Add 40mL distill water after 1stround
Add 150mLsodium citrate buffer after the 2nd round.
Let stand at room temperature in the buffer - 0h 20m 0s
Rinse in 1X TBS with Tween (TBST) – for 0h 5m 0s at -100Room temperature.
Immunostaining
Do not allow tissues to dry at any time during the staining procedure.
Apply a universal protein block (5% goat serum in TBST) – for 0h 30m 0s at -100Room temperature.
Drain protein block from slides, and apply diluted primary antibody overnight at 4°C.
Rinse slides in 1X TBST 3 times - 0h 5m 0s each at -100Room temperature.
Incubate secondary antibody (Conc - 1:400) - 0h 30m 0s at 37°C .
Rinse slides 1X TBST 3 times – 0h 5m 0s each at -100Room temperature
Mount the slides with ProLong Gold antifade
