Goblet cells in the PAS reaction,
Introduction,
In 'normal' histological staining, dye molecules are more or less bound to tissue and cell components.
In histochemical staining, a chemical reaction takes place between tissue and cell components with applied chemicals which results in a specific colour reaction or after which a specific dye can stain a specific component.
For example, lipids (fats) can be detected with the dye Sudan, Iron (II) with the Turnbull-blue reaction, Iron (III) with the Prusian-blue or Berliner-blue reaction, Nucleic acids (DNA) with the 'Feulgen' reaction and polysaccharides (carbohydrates) become visible with the PAS staining.
With enzyme histochemistry certain enzymes[2] can be made visible. Acid- and alkaline phosphatase[2] can be detected, oxidase and peroxidase, esterase and cholinesterase et cetera.
In Immunohistochemistry, cell components (antigens[2]) are detected with antibodies.
In-situ hybridization is a technique that makes it possible to localize specific nucleotide sequences in microscopic preparations.
In Lectin[2] histochemistry, certain sequences of sugar groups, such as those found in cell surface glycoproteins, can be bound by a specific lectin that has been pre-marked with a marker substance.
Most of the histochemical processing is almost impossible for hobby histology because it requires a lot of laboratory equipment and considerable financial investment.
However, histochemical reactions and staining can be carried out fairly easily and without a lot of expensive equipment and materials.
The purpose of this preparation is to detect polysaccharides in the goblet cells (mucus cup cells) located in the jejunum (middle part of the small intestine).
The histochemical staining for this purpose is called the: PAS staining (Periodic Acid Schiff).
The reaction is based on the oxidative action of periodic acid (HIO4) on the 1.2-glycol groups in the glucose residues, as shown in the reaction equation[1].
The resulting aldehyde[2] groups then react with Schiff's reagent to form an insoluble complex with purple-red colour[1].
The reagent can be purchased ready to use but has a relatively short lifetime (≈ 1 year depending on temperature and amount of light). It is better to always make a small amount yourself. This can be done quite easily and is described here.
For this purpose a piece of Jejunum has been prepared in paraffin. Also a piece of colon (large intestine) was prepared in Technovit to see if the mucus goblet cells there also react PAS positive. For orientation an overview of a piece of jejunum in a HE staining has been placed on the right. Click on the image for an enlargement.
1 = intestinal lumen;
2 = kerck ring folds (plicae circulares);
3 = villi (villi) with crypts (arrows) and villi (arrowheads);
4 = submucosa;
5 = muscularis mucosae with smooth muscle;
6 = blood vessels.
The followed PAS staining protocol originates from Stainsfile, the internet resource for histotechnologists[3]:
- Bring paraffin coupes by hydration into aqueous environment;
- Oxidize in periodic acid (1%), 25min;
- Rinse in tap water;
- Rinse in Aqua dest (AD);
- Apply Schiff's reagent and allow 25 to react;
- Rinse AD;
- Rinse in tap water (this step fortifies the pair/purple staining);
- Counter staining with haematoxylin (with haematoxylin is normally stained ≈ 5min but here no longer than 2-3min because otherwise the core staining will dominate too much. It is only for orientation here);
- Rinse AD;
- Blues in tap water;
- Rinse AD;
- Dehydrate and cover with e.g. Depex.
The images will show that: Goblet cells in the jejunum and colon of a mouse are PAS positive.
Click on the images,
References:
[1] Junqueira L.C. en Carneiro J. (2004, tiende druk), Functionele histologie, Maarssen. Uitgeverij Elsevier. Chapter 2, page. 31-32, 'Histochemie en cytochemie'.
[3] Stainsfile, the internet resource for histotechnologists, https://stainsfile.info/stain/stainindex.htm