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6. Preparation of 4% paraformaldehyde (PFA) in 0.1 M phos-
phate buffer (PB) solution
Materials
(a) Paraformaldehyde (PFA) powder
(b) Na2HPO4 dihydrate
(c) NaH2PO4 dihydrate
(d) Distilled water
(e) Stirring hotplate
(f) Filter paper
(g) NaOH
(h) pH meter
(i) Pyrex bottles and beakers
Procedure
Prepare 1 L of 0.1 M PB, pH 7.4.
Wear protective eye goggles, respirator mask, and appropriate
gloves during the whole solution preparation process.
(a) Put the PB solution in a flask supporting hot temperature.
(b) Place flask on top of the stirring hotplate inside the hood.
(c) Set the temperature of the hotplate to about 60°C and
check the solution temperature with thermometer. You
want to heat the solution to 55-60°C.
(d) Add PFA powder (40 g) carefully because it is volatile. Stir
the solution at mean speed. Inspect regularly to avoid
overheating and consequent spilling.
(e) Do not leave while your solution is on the heat.
(f) When the PFA has dissolved and the solution is clear,
switch off the heat but leave to stir: do not handle for safety
reasons. Allow to cool.
(g) Use filter paper to filter the whole solution when cooler.
(h) Then put the sensor of a pH meter in the solution with
slight stirring. Add NaOH as necessary to get a pH of 7.4.
(i) Transfer the fixative to a 4°C refrigerator. Label appropri-
ately and date. PFA 4% in 0.1 M PB can be store for 1
week before use in the refrigerator and for more than a
month at −20°C. If you need huge quantity you can fro-
zen 0.2 M 8%5 PFA solution and dissolve it into distilled
water when you are ready to use it.
7. The use of a peristaltic pump is convenient but not the best
choice as it does not respect the perfusion pressure and may
induce vessel damages (collapse or rupture).
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