Membrane alterations in cerebral cortex when using PIPES buffer

Summary

When used for vascular perfusion of brain, 0.1m PIPES-buffered 3% glutaraldehyde resulted in the formation of expanded, vesicle-filled cell processes limited by multiple membrane layers. These structures, termed multivesicular myelin figures and interpreted as artefacts, were most common in layer 2 of the cerebral cortex. When cacodylate or phosphate buffer was used instead of PIPES buffer in the primary fixative, such structures were not seen. The use of a more concentrated initial aldehyde fixative, PIPES-buffered, markedly reduced the size and numbers of these artefacts when compared to PIPES-buffered 3% glutaraldehyde only. Slowing the initial perfusion rate increased the size and frequency of occurrence of multivesicular myelin figures with PIPES buffer when compared to optimum perfusions. Prolonged initial exposure to PIPES buffer by using it to wash out the blood and then perfusing with fixative 5 min later did not increase the number or size of multivesicular myelin figures but did reduce the multivesicular nature of the artefacts. We suggest that the non-toxic nature of PIPES buffer allowed the formation of these membranous artefacts, while phosphate and cacodylate interfered with the cellular activity during the process of fixation.