Quantitative Assessment of Cerebral Basement Membranes Using Electron Microscopy

Methods Mol Biol. 2017;1559:367-375. doi: 10.1007/978-1-4939-6786-5_25.

Abstract

In this chapter we describe in detail the tissue processing techniques we employ for the study of cerebral tissue by transmission electron microscopy (TEM). In particular, we explain a technique that enables quantification of changes in cerebral basement membranes at the ultrastructural level. This is significant, as age related pathological conditions affecting the brain are often accompanied by ultrastructural changes in the cerebral vasculature.Briefly, experimental mice are fixed by perfusion and their brains removed. Brains are then vibratomed into 100 μm slices with regions of interest microdissected and processed for TEM following a protocol optimized for the preservation of cerebral tissue. Changes in the thickness of cerebral basement membranes are then quantified using novel software. Some prior knowledge of general TEM specimen preparation and sectioning will be useful when performing this protocol.

Keywords: Basement membrane; Dehydration; Electron micrograph; Fixation; Microtomy; Resin embedding; Tissue processing; Transmission electron microscopy; Ultra-thin sectioning.

MeSH terms

  • Animals
  • Basement Membrane / blood supply
  • Basement Membrane / ultrastructure*
  • Cerebral Cortex / blood supply
  • Cerebral Cortex / ultrastructure*
  • Desiccation / methods
  • Formaldehyde / chemistry
  • Glutaral / chemistry
  • Mice
  • Microscopy, Electron, Transmission / methods*
  • Microtomy / instrumentation
  • Microtomy / methods
  • Tissue Embedding / methods
  • Tissue Fixation / methods

Substances

  • Formaldehyde
  • Glutaral