1. Remove media from on top of gel carefully
  2. Remove media surrounding insert
  3. Rinse gel with PBS twice
  4. Fix with 3% paraformaldehyde (PFA) for 15 minutes at room temperature
  5. Following fixation wash gel with PBS
  6. Incubate gel with immunocytochemistry (ICC) blocking buffer for 30 minutes
  7. Remove ICC blocking buffer carefully
  8. Incubate gel in ICC block solution containing the primary antibody (rabbit polyclonal antibody to collagen type I/II for 1 h at room temperature
  9. Remove the non-bound primary antibody and wash three times using PBS
  10. Add TRITC-conjugated secondary antibody (goat anti-rabbit IgG-Alexa FluorÒ 594) for 1 h in the dark
  11. Remove the non-bound secondary antibody from gel and wash five times with PBS
  12. Mount gel onto the glass slide and add ProlongTM Gold anti- fade reagent with DAPI
  13. View gel under the confocal microscope