Fixation techniques for immunonfluorescence
Fixation techniques for immunonfluorescence
Contributed by Richard Gomer.
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
-
Methanol
- (can also try this at -20 degrees C)
-
- 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.
- 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.
- Without removing the HL-5, put 2 ml of PBM into the well. Gently swirl and remove.
- Add 1.5 ml of 9 ml PBM / 1 ml 37% formaldehyde.
- Swirl gently and fix for 30 minutes (we used to fix for 10 minutes, and this is not enough time).
- 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
- Dilute antibody in the above solution, don't spin, and add to coverslip for 1-2 hours
- 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
- 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
- Fix 30 min RT in above
- Postfix : 70% EtOH
- Wash in PBS/ 0.75% glycine
- Wash & stain PBS/ BSA/ gelatin
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