Fixatives are solutions of substances for preserving the morphological features of tissues similar to the alive features. They allow the processing of biological samples through histological techniques, minimizing the degradation of tissue structures. There are many fixative solutions that are chosen regarding the sample and the feature of the tissue to be studied.
PLP (periodate, lysine, paraformaldehyde) fixative solution is recommended for immunostaining, since it preserves antigens. Carbohydrates are fixed, since the fixative contains peryodate that oxidizes carnohydrates to form aldehydes and lysine forms bridges between them, thus cross-linking the molecules. Paraformaldehyde (formaldehyde when solved) is used at low concentration to estabilize the tissue. PLP is also a good fixative for electron microscopy, since it preserve membranes, so that the tissue can be processed for electron microscopy.
Solution A: add sodium phosphate dibasic 0.1 M to a solution containing lysine-HCl 0.2 M until the pH is 7.4. The resulting solution is diluted in buffer phosphate 0.1 M, pH 7.4 to get a final lysine-HCl concentration of 0.1 M.
Solution B: 8 % of paraformaldehyde in destilled H2O .
Fixative solution: mix 3 parts of solution A with 1 part of solution B, and add solid sodium metaperiodate to get a final concentration of 0.01 M of sodium metaperiodate.
Lysine-HCl: 0.2 M of Lysine-HCl in distilled water H2O
Sodium phosphate dibasic: 0.1 M of sodium phosphate dibasic in distilled H2O.
Sodium buffer phosphate: 0.1 M od sodium buffer phosphate in distilled H2O.
Paraformaldehyde: 8 % of paraformaldehyde in distilled H2O.
It is important to add the products and solutions in the order described above.
Dibasic sodium phosphate (Na2HPO4)
Sodium phosphate buffer
Sodium metaperiodate (NaI04)
Magnetic stirrer/heat plate