Peroxidase/DAB protocol

Leave sections in 0.125Molarity (M) phosphate buffer (PB) at 60°C

Dilute 0,4M PB . Store in refrigerator for up to 1 week.

100 ml: 31,25 ml0.4Molarity (M) PB + 68,75 ml H2O ₂O

Rinse sections 2 x 10min in PB

Rinse sections 1 x 10 min in 0,5% T BS-Tx

Tris : 3,03 gram/500 mlH₂O

NaCl : 4,48 gram/500 mlH₂O

Triton-X-100 : 2,5 ml/500 mlH₂O

Use HCl to adjust the 8. Store in refrigerator for up to one week.

Incubate with 10% goat serum in TBS-Tx

Incubate with primary antibody ( AT8; MAP2) 1:1000 in TBS-Tx with 5% goat serum,overnight in refrigerator 4°C

Rinse 3 x 10 min in TBS-Tx

DAB

Dissolve 1 tablet (10 mg) in 15 ml Tris-HCl by leaving it on a stirrer with heat (max 50°C) for about 2 hours. Add 12 µL H2O2 ₂O₂just before use and filtrate.

For dissolving the DAB use a brain cup (or any other cup you can throw away)

For filtrating the DAB use a disposable syringe with filter.

Incubate with secondary antibody, biotinylated goat anti-mouse, 1:500 in TBS-Tx with 5% goat serum, in room temperature

From the ABC -kit, put 1 drop of solution A and 1 drop of solution B in 5 mL TBS-Tx . Mix well and leave on the bench for 30 min before use.

Rinse sections 3 x 10 min in TBS-Tx

Incubate with ABC in room temperature

Rinse 3 x 10 min in TBS-Tx

Rinse 2 x 5 min in Tris-HCl

Incubate with DAB

Rinse 2 x 5 min in Tris-HCl

Mount sections in Tris-HCl on Superfrost slides and let them dry

Coverslip with Xylene and Entellan