Protocol: Immunofluorescence on chemically fixed cells

On-chip chemical fixation and immunofluorescence of HER2 receptor of breast cancer cell membrane

Materials

  • Cell medium
  • 1X PBS (also used as mounting medium)
  • Blocking buffer: NH4Cl 50 mM; BSA 0.5% in PBS
  • BB700 Mouse Anti-Human Her2/Neu (anti-HER2 Ab, 1:100 dilution in blocking buffer)
  • Mineral oil for cell culture
  • Stereomicroscope

Outline_protocol.png

Cell culture on VersaLive

Follow the protocol relative to the cell culture on VersaLive.

On-chip chemical fixation

  1. Empty all ports
  2. PBS washing (1x), 20 μL in all ports
  3. 10 min perfusion of PFA 4% in ports #1 to #5 (leave ports A and B empty)
  4. PBS washing (2x), 20 μL in all ports
  5. 5 min perfusion PBS washing, 20 μL in ports #1 to #5 (leave ports A and B empty)
  6. 30 min perfusion blocking buffer, 20 μL in ports #1 to #5 (leave ports A and B empty)
  7. Remove blocking buffer, fill all ports with 20 μL PBS
  8. [Optional pause point] Add 2.5 μL mineral oil in all ports to prevent evaporation, store at 4˚C

On-chip immunostaining

  1. 10 min perfusion anti-HER2 Ab, 20 μL in ports #1 to #5 (leave ports A and B empty)
  2. PBS washing (2x), 20 μL in all ports
  3. Mounting medium: 20 μL PBS in all ports + 2.5 μL mineral oil to prevent evaporation
  4. Store at 4˚C