Fixation techniques for immunonfluorescence

For new antibodies I suggest trying a variety of fixation techniques, as some antigens work with formaldehyde, others only work with ethanol fixation, etc. Here is a list of fixatives.

Fixatives (try 10 min and 30 min at room temp unless otherwise indicated)

• Ethanol

  • 95% ethanol

• Methanol

  • 100% Methanol
(can also try this at -20 degrees C)

• Fix from Pang, Lee & Knecht 1998

  • 9.38 ml HL5 (I used PBM)
  • 2 µl Triton X-100; mix, then add
  • 540 µl 37% formaldehyde
  • 80 µl 25% glutaraldehyde
This is great with phalloidin added in

• Low osmolarity fix

  • 9.4 ml water
  • 0.6 ml 37% formaldehyde
then open membranes with either (try both) 1%NP-40 in PBD or 95% ethanol

• AZF

  • 0.25 g Zinc chloride
  • 9.3 ml water; dissolve, then add
  • 1.5 ml formaldehyde
  • 95 µl acetic acid
bring to 10 ml with water

• Zinc Formalin

  • 0.1 g zinc chloride
  • 9 ml water; dissolve, then add
  • 1 ml formaldehyde

• Boiun

  • 0.75 ml saturated picric acid (keep picric acid in solution, dry picric acid can explode)
  • 0.25 ml formaldehyde
  • 50 µl acetic acid

• Boiun- Duboscq

  • 1.2 ml 95% ethanol
  • 0.3 ml water
  • 0.6 ml formaldehyde
  • 150 µl acetic acid
  • 10 µl picric acid

• Altmann

  • 0.03 g chromium potasium sulfate
  • 0.3 ml formaldehyde
  • 20 µl acetic acid
  • 2.4 ml water

• Bill Deery's new & improved cell fixation protocol

  Everything at room temperature and everything is done in the dish until the second Ab.
  
  1. Put 300 µl of cells in HL-5 on a glass coverslip in a 35 mm dish or multiwell plate for 15-20 min, so that they adhere and flatten.
  2. Without removing the HL-5, put 2 ml of PBM into the well. Gently swirl and remove.
  3. Add 1.5 ml of 9 ml PBM / 1 ml 37% formaldehyde.
  4. Swirl gently and fix for 30 minutes (we used to fix for 10 minutes, and this is not enough time).
  5. Permeabilize & block with T50BS/ 5%nonfat dry milk/ 0.25% Tween-20 for 15 minutes, remove
  T50BS is
  • 50 mM Tris/HCl pH 7.9
  • 100 to 300 mM NaCl (300 mM for reducing background)
  • 10 mM NaN3
  6. Dilute antibody in the above solution, don't spin, and add to coverslip for 1-2 hours
  7. Wash with the above solution 2 times 5 min each, then wash with Ab2 solution :
     • T20BS*
     • 1% BSA
     • 0.25% Tween-20
     *T20BS is TBS with 20 mM Tris HCl pH 7.5 not 7.9
     
  8. Then incubate Ab2 in T20BS on a cork in a humid box and wash in T20BS.

• Cardelli improvement:

  Grow cells on the coverslips! put cs into a dish, cover with HL5, add a drop of log phase cells, mix, and let sit 24 hours. Then gently fix as above.

• Gerisch Picric acid

  Kreitmeier et al 1995
  • 15% (vol/vol) sat'd picric acid in water
  • 1-2 % paraformaldehyde
  • 10 mM PIPES-HCl pH 6.0
  
  
  1. Fix 30 min RT in above
  2. Postfix : 70% EtOH
  3. Wash in PBS/ 0.75% glycine
  4. Wash & stain PBS/ BSA/ gelatin

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