2. Basic stages in the preparation of a permanent histological preparation. Hematoxylin-eosin staining.
Walks through the stages of making a permanent paraffin histological preparation: collecting and fixing small tissue pieces, decalcifying hard tissue, dehydrating and embedding, sectioning and mounting, then staining and sealing. Includes a fixative comparison table, a table contrasting hematoxylin and eosin, the H&E staining steps, a table of other stains, and a brief note on frozen sections.
Topic 2 of 127 in the MU Sofia syllabus for Cytology (Dental Medicine, Year 1). The free AceMed note covers it in 11 sections, about 1,026 words (9 min of reading), with 14 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
Collection of material for microscopic examinations. Fixation.
Slide Preparation for Microscopic Examination
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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