Staining Protocol

The following staining protocol was used for all dual color images available on this site.

Basic Reagents

• 1x PBS
• PBT (1 X PBS, 0.3% Triton X-100)
• 4% Paraformaldehyde
• Primary Antibodies
  • rabbit anti-GFP (Invitrogen, 1:1000 dilution)
  • mouse anti-brp (NC82 Developmental Studies Hybridoma Bank, 1:20 dilution)
• Secondary Antibodies
• goat anti-rabbit 488 (Invitrogen, 1:400 dilution)
• goat anti-mouse 546 (Invitrogen, 1:400 dilution)
• Vectashield/Slides etc.

Day 1

1. Dissect in PBS-solution (quickly)
2. Fix in 4% Paraformaldehyde (on Ice) for 60 minutes
3. Wash in PBS once for at least 5 mins at RT
4. Wash in PBT three times for at least 20 minutes each time at RT
5. Incubate in primary antibodies : PBT overnight at 4°C

Day 2

1. Wash in PBT three times for at least 20 minutes each time at RT
2. Incubate in secondary antibodies : PBT overnight at 4°C

Day 3-5

1. Wash in PBT for at least 2 days
2. Mount on slide under Vectashield