Protocol
Day 1
Day 2
Buffers
1X PBS, 0.1% Tween20 (PBT) [50 mL]
Blocking solution (1X PBS, 0.2% TritonX/Tween20, 2% normal goat serum ) [1 mL]
Day 1
- Allow sections to dry for 2 hr at RT.
- Draw boundary with PAP pen.
- Rehydrate slides with 500 uL PBT, 30 min RT.
- Apply 300 uL blocking solution, 1–2 hr RT.
- Apply primary antibody diluted in blocking solution and incubate at 4 C O/N in humid chamber.
Day 2
- Remove primary antibody (can be reused 2x) and 4x wash in 300 uL PBT, 10 min RT.
- Apply secondary antibody in blocking solution, 2–4 hr RT or 1–2 hr 37 C.
- Cy2 1:100, Cy3 1:500, Cy5 1:200
- 4x wash in 300 uL PBT, 10 min RT.
- Hoechst (10 mg/mL) in ddH2O, 15 min RT.
- 2x wash 300 uL PBS, 5 min RT.
- Apply 40 uL 80% glycerol, place coverslip on and seal with nail polish.
Buffers
1X PBS, 0.1% Tween20 (PBT) [50 mL]
- 5 ml 10X PBS
- 50 uL Tween20
- 45 mL ddH2O
Blocking solution (1X PBS, 0.2% TritonX/Tween20, 2% normal goat serum ) [1 mL]
- 977 uL 1X PBS
- 2 uL TritonX-100 (for day 1) or Tween20 (for day 2)
- 20 uL normal goat serum