Formaldehyde is a widely used fixative in most histological techniques. It is the main component of several fixatives, such as Bouin, FAA, and PLP, or it can be used alone as buffered 4 % formaldehyde. It is a good practice to prepare the fixative just before the fixation to prevent oxidative processes. Nowadays, it is often prepared from paraformaldehyde, which a polymer (bought as powder) that can be readily dissolved to get the working solution.
Procedure
4 % buffered formaldehyde
4 g paraformaldheyde
25 ml phosphate bupher 0.4 M, pH 7.4
1 o 2 drots of sodium hydroxide 10 N
75 ml distilled water
How to. For 100 ml of fixative.
1. Heat 60-70 ml of distilled water at 60 ºC in a flask containing a magnetic stirrer.
2. Add 4 g paraformaldehyde.
3. Add 2 or 3 drops of sodium hydroxyde 10 N.
4. Stirr until the solution gets clear.
5. Filter the solution.
6. Add 25 ml of phosphate buffer 0.4 M pH, 7.4
7. Add distilled water to get 100 ml of total volume.
8. Cold de solution at 4-10 ºC.
Result: 100 ml of 4 % formaldehyde in phosphate buffer 0.1, M, pH 7.4.
Notes
Formaldehyde may preserve lipids but it is more effective with calcium ions added to the solution.
Formaldehyde does not bind carbohydrates, and therefore glycogen and glycosaminoglycans can be lost.
For electron microscopy, foramaldehyde is combined with glutaraldehyde (0.5 - 3 %). Picric acid can be included as well.
The quality of fixation can also be improved by adding lysin, cyclohexylamine or phenol. They can be used instead of glutaraldehyde.
Diferent paraformaldehyde-containing fixatives can be used sequentially. For example, an initial and short fixation with 4 % of formaldehyde and 2 % phenoal at pH 7 can be followed with a longer fixation with the same solution but adjusted to pH 5.
Products
Parafolmaldehyde
Sodium hydroxide10 N
Phoshate buffer 0.4 M pH 7.4
Distilled water
Labware
Test tube
Funnel
Fiter paper
Magnetic stirrer
Heater-stirring plate