Reagents

• Blocking Buffer: PBS with 0.025% Tween-20 and 5% Normal Goat Serum
• Wash/Incubation Buffer: PBS with 0.025% Tween-20 and 0.2% Normal Goat Serum

Sample Preparation

• Sample is fixed in PFA overnight at RT, then parafin embedded and sectioned by histology
• Remove Parafin by 3x 4 minute washes in Xylenes
• Remove Xylenes by 3x 2 minute washes in 100% EtOH, then 2 minute washes in 95%, 70% Ethanol
• Put into PBS, never allow sections to dry - use humidified chamber
• Mark around section with PAP pen or grease pencil

Blocking

• Block at room temperature for 30-60 minutes in Blocking buffer

Primary Antibody

• Dilute 1:10 in Wash/Incubation buffer
• Incubate on slide 30 minutes at 37°C, 90 minutes at RT, or o/n at 4°C

Wash

• Siphon off antibody, and wash 5x 4 minutes with 500µl Wash/Incubation Buffer at RT

Secondary Antibody

• Use CY-3 at 1:200, 60 minutes RT in Wash/Incubation buffer, in dark

Wash

• Siphon off antibody, and wash 5x 4 minutes with 500µl Wash/Incubation Buffer at RT

Hoechst Stain Nuclei

• Stain for 2 minutes with Hoechst 33342 (or 33358) in Wash/Incubation buffer at RT in dark
• Wash 1x 4 minutes with Wash/Incubation Buffer
• Add 1 drop mounting medium (VectaShield) and cover glass