Formaldehyde Fixation

Fixative stock: 37% Formaldehyde: small bottle under chemical hood in prep lab.
TritonX-100: 10% stock, in hood in prep lab

To make 10 mL of fixative:

To a 15 mL conical plastic tube add:
1mL of 37% formaldehyde
0.5 mL of 10% triton
1 mL of 10x PBS
dH2O to 10 ml mark (8.5 mL)

Make in fume hood

To fix the cells:

  • Gently rinse cells 2X in warm PBS (remove medium, replace with PBS,
    remove PBS, replace with fresh PBS, remove again, replace). Leave
    cells in this PBS—until you add the fixative).
  • removing final PBS rinse and add about 1 mL of fixative to the cells; fix cells in hood for 10 mins.
  • Rinse cells by dunking each coverslip in 3 beakers of PBS-Tween-Azide, 10 X per beaker.
  • Place coverslip back in dish with fresh PBS-Tween-Azide
  • If you are not staining immediately, store cells in PBS-Tween-Azide in fridge.

    • Important things to remember

    Wear gloves and be very careful of the fixatives!!!
    Remember which side of the coverslip has the cells.
    Discard used fixative into the appropriate hazardous waste container in
    the chemical hood in the main lab.

    Paraformaldehyde fixation

    Fixative Stocks:
    32% Paraformaldehyde: bottle under chemical hood in main lab.
    Detergent: 10% triton X 100

    To make 10 mls of fixative:
    Paraformaldehyde: 1 mL stock
    10% triton: 0.5 ml
    10X PBS -/- 1 mL
    add water make 10 mL (8.5 mL)

    Use same method as Formaldehyde fixation.