Fixatives

Fixatives kdorfman Tue, 06/16/2009 - 17:53

How to fix cells and tissues

Fixing adherent cells

Fixing adherent cells kdorfman Thu, 05/21/2020 - 17:39

To fix the cells grown on a coverslip

  • Work in a fume hood
  • Gently rinse cells 2X in warm PBS (remove medium, add PBS, repeat)
  • Leave cells in this PBSā€”until you add the fixative.
  • Remove final PBS rinse, 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.

Remember:

  • Work in the fume hood
  • Wear gloves!
  • Keep track of which side of the coverslip the cells are on
  • Discard used fixative in the appropriate hazardous waste container in the hood.

Formaldehyde fix

Formaldehyde fix kdorfman Thu, 07/05/2012 - 20:55

Make in fume hood 3.7% formaldehyde
0.5% Triton X
in PBS

Formaldehyde stock: 37%
Triton X stock: 10%

To make mL fixative
add: mL 37% formaldehyde
add: mL 10X PBS
and: mL 10% Triton-X

To fix the adherent cells (still in hood):

Methanol fix

Methanol fix kdorfman Tue, 07/24/2012 - 17:05

100% methanol at ice temperature

10 min

No permeabilization step needed.

Wash with PBS afterwards.

The methanol fixation is an easy method; however, it frequently solubilizes and removes membrane bound antigens. By a simple precipitation of the protein, methanol only provides low structural preservation.

PFA 5% for fish

PFA 5% for fish kdorfman Wed, 10/02/2019 - 16:56

5% paraformaldehyde
in PBS

Paraformaldehyde stock: 32%

To make mL fixative
at: % paraformaldehyde
add: mL 32% paraformaldehyde
add: mL 10X PBS

Paraformaldehyde Fix

Paraformaldehyde Fix kdorfman Sun, 10/27/2013 - 19:32

3.7% paraformaldehyde
in PBS

Paraformaldehyde stock: 37%
Triton X stock: 10%

EMS RT 15714

To make mL fixative
add: mL 37% paraformaldehyde
add: mL 10X PBS

To fix the adherent cells (still in hood):

Paraformaldehyde from powder

Paraformaldehyde from powder kdorfman Wed, 03/03/2021 - 20:12
to make: mL
at a concentration of: percent
start with: mL 10X PBS
then add: mL water
add and stir: g paraformaldehyde powder

Stir, heat to ~60C

Add NaOH until it clears, then bring to final volume

Paraglut

Paraglut kdorfman Mon, 09/24/2012 - 16:12

3.2% paraformaldehyde
0.1% glutaraldehyde
0.5% Triton X-100

in PBS

To make mL fixative
add: mL 37% paraformaldehyde
add: mL 10X PBS
and: mL 10% Triton-X
add: mL 1glutaraldehyde

Bring to final volume with distilled water.

To fix adherent cells (still in hood):