The Influence of Buffer Nature on Scanning Electron Microscopy (SEM) of Glutaraldehyde-Fixed Tissues
Tissue and cell preparations for scanning electron microscopy (SEM), fixed with glutaraldehyde (G CHO) and buffered in Na-cacodylate or piperazine-NN’-bis (ethanesulfonic acid) (PIPES), exhibit distinct morphologies. This study describes the impact of these buffering agents in tissue fixation and washing solutions on the appearance in scanning electron microscopy. Biochemical determinations of lipid retention were conducted on frog and chicken embryos fixed for 24 hours with 3% G CHO in 0.03 M 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 could enhance the sensitivity to buffer effects becoming apparent. The use of comparable small and uniform embryo sizes minimized fixation quality differences due to penetration. Lipids recovered from homogenized tissue after treatment with chloroform/methanol/water (1:2.1:1, v/v) were considered preserved. The extraction results, showing a significant reduction in lipid losses when using PIPES buffer, were considered in light of the observed morphological differences in SEM and transmission electron microscopy (TEM).