Embedding, sectioning, immunocytochemical and stereological methods that optimise research on the lesioned adult rat...

Numerous methods have been developed to embed, section and immunocytochemically label nervous tissue and the method chosen depends upon numerous factors. However, many of these methods have technical drawbacks that make them difficult to use in studies using injured/lesioned tissue. We present here, methods for embedding, sectioning and immunocytochemically labelling lesioned adult spinal cord tissue at the light microscope level. We have developed a novel, gelatine-embedding technique for vibratome sectioning which overcomes many of the difficulties encountered with lesioned tissue. Individualised immunocytochemical protocols have also been developed for the antibodies GFAP (to label astrocytes), MBP (to label myelin) and CNP-ase (to label oligodendrocytes). Sequential pre-treatment with proteinase-K, methanol and sodium borohydride achieved optimal GFAP localisation. MBP and CNP-ase were optimally localised after sequential pre-treatment with proteinase-K (at different concentrations) and sodium borohydride. Methanol pre-treatment resulted in a loss of immunoreactivity for these latter two antibodies. Each protocol achieved full immunocytochemical penetration throughout 40 microns vibratome sections. These techniques enable the new unbiased stereological tools (that is, the Cavalieri and optical disector principles) to be utilised.