Immunofluorescence Assay for DRG Cultures

Cell Fixation

  1. Remove culture media from wells
  2. Add 500ul 4% Paraformaldehyde to each well in a 24 well plate for 20 minutes at RT
  3. 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

  1. Dilute primary antibody in PBST or blocking buffer
  2. Add 500 ul of primary antibody solution for 1 hour at RT

Secondary Antibody

  1. Wash the cells 3x with 1x PBST for 5 minutes
  2. Dilute secondary antibody (using different channels) and DAPI in PBST or blocking buffer
  3. Add 500 ul of secondary solution for 1 hour at RT
  4. Wash the cells 3x with 1x PBS for 5 minutes
  5. Protect from light with foil and store in 4ºC