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PFA fixation (Hippocampal Neurons in culture)

The first steps are done at 37C to prevent any temperature stress to the cells before they
are fixed. It also helps in preserving the cell structure (microtubules and others). You can
wash the cells before fixing them, in that case you have to warm up your extracellular
solution to 37C before use. If you don’t wash them go directly to the fixation step.

Pre Washing

Take out the plate from the incubator and transfer the coverslips right away to a fresh
plate or dish containing pre warmed extracellular solution.

Fixation

Take out the plate from the incubator and transfer the coverslips right away to a fresh
plate or dish containing pre warmed PFA solution, or if you washed the coverslips first,
transfer them right away to a fresh plate or dish containing pre warmed PFA solution. use
500 μl /well for a 24 well plate; 1mL/well for a 12 well plate. At this point, the plate can
stay at RT and the temperature will go down while the cells are fixing.
Incubate at room temperature for 10 minutes.

Post fixation Washing

Transfer the coverslips right away to a fresh plate or dish containing 20 mM PBS pH 7.4
(once the cells are fixed, it’s OK to use PBS w/o calcium). Use 1 ml / well for a 24 well
plate; 2mL/well for a 12 well plate
Incubate for 5-10 minutes at RT.
repeat once

Then for the final incubation, take some PBS and add 0.1 M glycine to it (this will
quench traces of PFA). Transfer the coverslips to that solution and incubate for 5-10
minutes if you are going to use the samples right away or leave them in the fridge for a
few days. They can also be mounted right away with Prolong Antifade reagent or
Mowiol.

Note: It is not advisable to use your solutions (PBS or PFA) for more then 1 coverslip, it
may yield differences in your coverslips. It is better to transfer the coverslips to a fresh
well for each new step then to aspirate the liquid. Aspiration may dry out the cells and
give crappy results.

Extracellular solution (neurons grown in neurobasal complete media)

(warm up solution at 37 degrees before use) note: make sure that this solution is OK to use in your experiment.

51 mL NaCl 2M stock
2.5mL KCl 2M stock
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10 ml Na-Hepes 1M pH 7.8 stock

10ml glucose 1M stock
1.2 ml CaCl2 1M stock
1 ml MgCl2 1M stock
dd H20 to 1L
osmolalily : ~230 mmol / kg
pH 7.3 – 7.4

PFA solution
(warm up solution at 37 degrees before use)

Example for 20mL:

- 5.0 mL PFA 16 % final : 4%
- 5.0 mL Sucrose 16 % (frigo) final : 4%
- 4.0 mL Phosphate buffer 0.5 M pH 7.4 final : 0.1M
- 5.6 mL ddH2O stérile
- 400 mL Na-EGTA 100 mM pH 8.0 final : 2 mM ***Not always necessary:
aldehydes can cause an increase of calcium in cells, the EGTA would prevent a
potential effect of that calcium on proteins.

20 mM PBS pH 7.4

80 ml of 500 mM Na-phosphate solution pH 7.5 (Sorensen)

150 ml 2M sodium chloride
H20 to 2L

500 mM Na-phosphate pH 7.5 solution (Sorensen)

sol. A: 500 mM sodium phosphate monobasic

sol. B : 500 mM sodium phosphate dibasic
Mix A and B to get pH = 7.5 ( about 1: 4)