Tissue sectioning for epifluorescence microscopy

Abstract

Specimens labelled with multiple fluorophores illuminating a variety of cellular targets, facilitated by developments in probes, data acquisition and analysis technologies, have extended the capabilities of anatomical investigations. However, the field has largely neglected development and consensus in optimal techniques of specimen preparation. The optical properties of the specimen largely determines study outcome in epifluorescence microscopy. Thus, we discuss tissue preparation protocols and their application in multiple anatomical investigations. The processing of tissues optimised for multiple studies are detailed. The conservation of cyto-architecture and antigenicity, reduced tissue autofluorescence, increased permeabilisation, and applications for thick tissue sections are examined. Multiple analytical assays of the same tissue sections for the cross correlation of multiple data sets are presented. The application of multiple labelling immunofluorescence, fluorescent in situ hybridisation and fluorescent histochemistry, in qualitative and quantitative analysis are considered. Keywords Anatomy; Frozen sections; Fluorescence histochemistry; Immunofluorescence; in situ hybridisation; Microscopy; Morphometry; Paraffin; Polyethylene glycol; Stereology; Tissue sectioning.