Immunofluorescence Assay for DRG Cultures
Cell Fixation
- Remove culture media from wells
- Add 500ul 4% Paraformaldehyde to each well in a 24 well plate for 20 minutes at RT
- Wash the cells 3x with 1x PBS for 5 minutes
Permeabilization
- Add 500ul of .2% TritonX-100 in PBS (PBST) (gently) for 10 minutes at RT
- For blocking buffer add 10% serum (goat/donkey) to PBST
Primary Antibody
- Dilute primary antibody in PBST or blocking buffer
- Add 500 ul of primary antibody solution for 1 hour at RT
Secondary Antibody
- Wash the cells 3x with 1x PBST for 5 minutes
- Dilute secondary antibody (using different channels) and DAPI in PBST or blocking buffer
- Add 500 ul of secondary solution for 1 hour at RT
- Wash the cells 3x with 1x PBS for 5 minutes
- Protect from light with foil and store in 4ºC