3. Basic stages in preparation of permanent histological preparation. H&E staining.
Walks through the ordered steps for making a permanent paraffin section: sampling and fixation, decalcification of hard tissue, dehydration, clearing and embedding, microtome sectioning and mounting, then staining and sealing. Covers how hematoxylin and eosin work and the steps of the H&E procedure, with tables comparing fixatives, the two dyes and several other stains. Also outlines frozen sections as a faster alternative.
Topic 3 of 97 in the MU Sofia syllabus for Cytology (Medicine, Year 1). The free AceMed note covers it in 12 sections, about 1,418 words (12 min of reading), with 12 flashcards.
In this note
Introduction
Taking the material
Fixation
Washing, and decalcification of hard tissues
4–6. Making a paraffin block
Sectioning and mounting
8–11. Staining and sealing
A quicker route: frozen sections
Hematoxylin–eosin (H&E) staining
Microscopy. Types of microscopes.
Other Methods
Summary of Key Points
Introduction
A permanent preparation is a thin, stained slice of tissue sealed under a coverslip, which can be kept and studied for years. Making one is a chain of steps in a fixed order, and each step leaves the tissue ready for the next. The routine method uses paraffin embedding and hematoxylin–eosin (H&E) staining.
Taking the material
Sources: a biopsy from a living patient, a surgical specimen, an autopsy, or experimental animals.
Pieces are kept small, no thicker than about 5 mm, so the fixative reaches the centre quickly.
Material is taken as soon as possible, with a sharp blade and without crushing or stretching. Otherwise autolysis (self-digestion by the cell's own lysosomal enzymes) and bacterial decay begin.
Fixation
Fixation keeps the tissue as close as possible to its living state. It stops autolysis and decay, kills microorganisms, hardens the tissue for cutting and makes its structures easier to stain. The fixative should be about 10–20 times the volume of the tissue.
Collecting the material and fixation have a note of their own with more detail.
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