Biointerfaces Between Cells and Substrates in Three Dimensions - Biointerfaces: Where Material Meets Biology - page 144

A broad range of histological procedures is available, using different
fixatives, chemical stains and antibodies. As the purpose of this chapter is to
merely highlight some of the general techniques to examine cells at the 3D
interface, only a brief example of a common processing procedure is de-
scribed. The first step in the process is to fix the cells on the 3D substrate
with the appropriate fixative. Examples include Bouin's fixative (preferred
for haematoxylin and eosin staining procedures) and paraformaldehyde
(usually preferred for immunocytochemistry applications). After fixation the
samples are then dehydrated through a series of ethanol gradients and then
placed into a clearing agent (e.g. Histo-Clear; National Diagnostics). Samples
are then embedded into paran wax and then sectioned into sections of
5-10 mm thickness before being placed onto a glass slide. Once the sample is
on the glass slide, a variety of different staining procedures can then be
performed to image cells, proteins and other biomolecules. Simple histology
stains like haematoxylin and eosin can be used to obtain valuable cross-
sections of cells in the 3D environments, such as the example from Alvetex s
in Figure 6.8. Alternatively, immunocytochemical procedures can be em-
ployed, where antigen specific antibodies coupled to a fluorophore or col-
orimetric stain (directly or indirectly) can be used to detect almost any cell
marker.

d n 3 r 4 n g | 3

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Figure 6.8 Histological analysis of HepG2 cells cultured on Alvetex s , using haema-
toxylin and eosin staining. In this example cell nuclei have been stained
dark blue (haematoxylin) and cell cytoplasms have been stained pink
(eosin); however, a range of alternative stains are available.
Reinnervate is acknowledged for supplying the image and allowing reprint
in this chapter. Scale bar ¼ 50 mm.