Fixative Recipes

Periodate-Lysine-Paraformaldehyde (PLP)

Reagents

A. Lysine HCl solution

Lysine HCl, 13.7 g
Na₂HPO₄.2H₂O (Sorenson’s salt), 1.8g
Distilled water, 475 ml

Method

Dissolve lysine HCl in 375 ml of distilled water.
Dissolve Sorenson’s salt in 100 ml of distilled water.
Add Sorenson’s salt solution to lysine solution until pH 7.4 is reached.
Discard any remaining Sorenson’s salt solution.

Reagents

B. Paraformaldehyde solution

Paraformaldehyde, 2.5 g
Distilled water, 200 ml
1 M NaOH, few drops

Method

Heat the distilled water (or, sometimes a 5% dextrose solution is used) to 60°C.
Add the paraformaldehyde, and a few drops of 1 M NaOH while stirring to clear the solution.
Leave to cool (refrigerate overnight if necessary).

Reagents

C. Final PLP solution

Lysine HCl solution (Solution A)
Paraformaldehyde solution (Solution B)
NaIO₄ (Sodium Periodate), 2.14 g

Method

Make up the final solution immediately before use by mixing together the Lysine HCl solution and, the paraformaldehyde solution, and then adding 2.14 g of NaIO₄.
Make up to 1 liter with 0.1 M phosphate buffer and, adjust the pH to 7.2.

Based on: McLean, I.W. and Nakane, P.K. (1974). Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J. Histochem. Cytochem. 22:1077-1083.

Zamboni’s Fixative

Reagents

Paraformaldehyde (from 16% stock solution), 125 ml
Picric acid (saturated aqueous), 150 ml
10 M NaOH, a few drops

Method

Combine paraformaldehyde and picric acid solution. Make up to 1 liter with 0.1 M phosphate buffer. Adjust to pH 7.3 using 10 M NaOH.

Based on: Stefanini, M. et al. (1967). Fixation of ejaculated spermatozoa for electron microscopy. Nature 216: 173-174.

Bouin’s Fluid (aqueous)

Reagents

Picric acid (saturated aqueous), 75 ml

Formalin (37-40% formaldehyde), 25 ml

Glacial acetic acid, 5 ml

Method

Combine all three to make up the fixative.

Based on: Hopwood, D. (1990). Fixation and fixatives. In Bancroft, J.D. et al. (eds). Theory and practice of histological techniques. Churchill Livingstone, New York, p21-42.