| | Protocol for Labeling of Neurons cells - immunohistochemistry - Take slides out from -20
- Let them dry at RT, not more then 10 min
- Use Pap pen (contains oily substance) to place a barrier around the sections
- Rehydrate slides with 1x PBS for 5 min
- Using suction dry around sections, but not sections directly
- Then wash with 1x PBS+ 0.2 NP-40 (or Triton) solution for 5 min, 3 times
- Then block with 1% Normal Donkey Serum (50 ul serum + 5 ml 1x PBS+ 0.2 NP-40) at least 30 min (40 min-1 hr)
- Apply primary antibodies ON (approx. 16 hr) at 4C, 200 ul is enough for 9 sections
NEUN is mouse antibody, general marker for neurons: You can use 594 or 568 nm secondary antimouse, but 594 is preferred; dilution is 1:500 in PBS for secondary antibody
for YPF, we have rabbit antibody: For the secondary antibody use 488nm antirabbit , dilution is 1:500 in PBS for secondary antibody - Wash slides with 1x PBS for 5 min, 3X
- Apply fluorescently labelled secondary antibodies, 1:500 dilution, 200 ul for one slide, and keep in humidity chamber for at least 1 hr up to 2 hrs.
- Remove sec ab
- Wash with 1x PBS for 5 min, 3X
- Remove PBS and apply 1-2 drops of VectaShield solution
- Place appropriately sized coverslip
- Dry excess solution by putting slides facing down onto tissue paper
- Seal with nail polish if you want to store them for an extended time
- Store in slide holder at 4C until microscopy
|
|