The influence of the buffer on maintenance of tissue lipid in specimens for scanning electron microscopy.

Abstract

Preparations of tissues and cells for scanning electron microscopy (SEM) fixed with glutaraldehyde (G CHO) buffered in either Na-cacodylate or Piperazine-N-N' bis (20-ethanol sulfonic acid) (PIPES) differ in their morphology. The influence of the nature of these buffers in the tissue fixing and washing solutions on appearance in scanning electron microscopy is described. Biochemical determinations of lipid retention were performed on frog and chick embryos fixed for 24 hours with 3% G CHO in either 0.03 and 0.1 M PIPES (ph. 7.3) or 0.1 M Na-cacodylate (ph. 7.3). Embryonic tissue was chosen for its relatively high lipid content and delicacy which may be expected to enhance the sensitivity at which buffer effects become apparent. The comparable small and uniform sizes of the embryos minimize differences in fixation quality due to penetration. Lipid, recovered from homogenized tissue after treatment with chloroform/methanol/water (1:2.1:1, v/v) was considered as retained. The extraction results, which showed a significant reduction of lipid losses when PIPES buffer was used, are taken to account for the morphological differences observed in SEM and transmission electron microscopy (TEM).