Science Gateway > Protocols > Cell Biology Protocols - Table of Contents
Star Republic: Guide for Biologists

Staining --- cells on slide

  • 1. Wash cells gently with PBS at room temperature.
  • 2. Fix with cold methanol (-20 C) for 5 minutes. See Fixation for other options.
  • 3. Fix in (-20 C) acetone for 2 minutes.
  • 4. Rinse 3 times with PBS.
  • 5. Cover cells with 100 ml properly diluted primary antibody in PBS. Cover with a coverslip. Incubate for 1 hour at 37 C or room temperature.
  • 6. Rinse 3X 10 minutes with PBS.

    Immunofluorescence

  • 7. Cover the cells with properly diluted secondary antibody couple to FITC or Texas-Red. Cover with a coverslip. Incubate 0.5-2 hours at room temperature or 4 C.
  • 8. Wash with PBS, 3 x 10 minutes.
  • 9. Mount and view under a fluorescence microscope.

    Immunoperoxidase

  • 7a. Cover the cells with properly diluted secondary antibody (biotinylated). Cover with a coverslip. Incubate 0.5-2 hours at room temperature or 4 C.
  • 8a. Wash with PBS, 3 x 3 minutes.
  • 9a. Apply Strepavidin/HRP and incubate at room temperature for 30 minutes.
  • 10a. Wash with PBS, 3 x 3 minutes.
  • 11a. Stain with DAB.
  • 12a. Wash with running tap water for 3 minutes.
  • 13a. Mount.