Fixation techniques for immunonfluorescence

Low osmolarity fix

• 9.4 ml water
• 0.6 ml 37% formaldehyde

AZF

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

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

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
6. T50BS is
   • 50 mM Tris/HCl pH 7.9
   • 100 to 300 mM NaCl (300 mM for reducing background)
   • 10 mM NaN3
7. Dilute antibody in the above solution, don't spin, and add to coverslip for 1-2 hours
8. 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
   
9. Then incubate Ab2 in T20BS on a cork in a humid box and wash in T20BS.

Cardelli improvement: