Immunohistochemical staining, vibratome sections

Incubate samples in 1 x PBS 3% H₂O₂, for 0h 10m 0s at Room temperature on the wobbler (to quench endogenous peroxidase activity) using 500µL / well.

Rinse with 0.1% (v/v).

Rinse with 0.1% (v/v) for 0h 5m 0s on wobbler.

Repeat: rinse with 0.1% (v/v) for 0h 5m 0s on wobbler.

Add 250µL (accordingly to the secondary Abs) 0h 40m 0s at Room temperature on wobbler.

Rinse with 0.1% (v/v).

Rinse with 0.1% (v/v) for 0h 5m 0s on wobbler.

Repeat: rinse with 0.1% (v/v) for 0h 5m 0s on wobbler.

Add 250µL for 0h 30m 0s at Room temperature on wobbler.

Rinse with 0.1% (v/v).

Rinse with 0.1% (v/v) for 0h 5m 0s on wobbler.

Repeat: rinse with 0.1% (v/v) for 0h 5m 0s on wobbler.

Add 250µL for 0h 30m 0s at Room temperature on wobbler.

Rinse with 0.1% (v/v).

Rinse with 0.1% (v/v) for 0h 5m 0s on wobbler.

Repeat: rinse with 0.1% (v/v) for 0h 5m 0s on wobbler.

Prepare DAB solution: 10mg for 25mL; dissolve and filter through 0.22 µm filter, add H₂O₂ just before use! Or Vector SG (TH).

(Add 1.4µL for 5mL).

Add 250µL (ON DAB BENCH!) . Allow reaction to proceed for a few minutes at Room temperature without wobbling .

Rinse with 0.1% (v/v).

Replace TBS Triton X-100 with PBS or 0.1% (v/v) in case sections are not to be mounted immediately. Store at 4°C.

Briefly rinse sections in ½ PBS + ½ AD and mount on gelatin coated microscopy slides. Allow to dry for 0h 30m 0s in flow or couple of hours on bench.

Dehydrate samples in 70% (v/v) for 0h 5m 0s.

Dehydrate samples in 90% (v/v) for 0h 5m 0s.

Dehydrate samples in 100% (v/v) for 0h 5m 0s.

Repeat: dehydrate samples in 100% (v/v) for 0h 5m 0s.

Replace ethanol with Histoclear II for 0h 5m 0s.

Mount coverslips on top of the slides with DPX and allow to dry 0h 30m 0s in the flow.

Press out bubbles next morning.