Introduction to Special Stains Dr Vivien Rolfe De

Introduction to Special Stains Dr Vivien Rolfe De Montfort University This is an Open Educational Resource (OER) that is globally available on the web Creative Commons BY SA

Summary �Why are special stains still important and relevant today? �What are some of the chemical principles behind these stains? �Some common examples that can be prepared in student laboratory teaching.

What are “special stains”? � H&E was first introduced in the 1870’s and the term special stain came to refer to any technique other than H&E used in the clinical environment. � Whilst the H&E stain is the most common staining method in hospital and research laboratories, it isn’t without its limitations. • H&E cannot visualize micro-organisms. • H&E is not good for distinguishing connective tissue and nerve tissue. • H&E cannot distinguish molecular basis of disease and immunohistochemistry might be preferred.

Six Special Stains!

1) Toluidine Blue �Tolonium Chloride �Useful blue cationic dye �Cheap and simple application

Neutrophils separated from blood sample Stain for 3 minutes in 0. 5% toluidine blue

Buccal cells

Distinguishing nerve tissue

2) Luxol Fast Blue �Immunohistochemical methods have advanced but are costly and reagents deteriorate quickly. �Special stains include silver methods (such as Gordon and Sweet’s), gold, or Luxol fast blues to stain myelin. �Other special stains identify nerve cells. The techniques are important for looking and neurodegeneration.

Distinguishing connective tissue Reticulin Collagen Fibrin

3) Gordon and Sweet’s �One of several silver methods for staining reticulin. �Tissue treated with potassium permanganate to enable the silver to bind. �Uses an ammoniacal silver solution. �What is reticulin? �Why is it important?

Gordon and Sweet’s (Low Power) Liver tissue with no counterstain Reticulin = black

Gordon and Sweet’s (High power) Liver counterstained with what? Reticulin = black Cytoplasm = pink

4) Van Gieson �Trichrome stain – producing 3 colours. �Anionic dye and a cationic counterstain. �Nuclear stain applied first such as Weigert’s haematoxlin. �Collagen stains red with acid fuchsine. �Cytoplasm including muscle stains yellow. �Washing in acidified water differentiates tissue producing two colours.

Van Giesen (High power) Bladder Collagen = red All other tissue including transitional epithelium = yellow

Van Giesen (Low power) Collagen = red Smooth muscle = yellow Epithelium = yellow Application? Might be used to localise tumours in the bladder to either the smooth muscle or connective tissue layers.

5) Martius scarlet blue stain �Trichrome stain. �Martius yellow and phosphotungstic acid. �Brilliant crystal scarlet. �Methyl blue. �What do the dyes stain?

Tongue (High power) Epithelium = red Collagen = blue Cytoplasm = red No visible yellow

Bladder (low power) Collagen = blue Erythrocytes and early fibrin = yellow Cytoplasm = pink

Adrenal gland (100 x oil immersion) Erythrocytes clearly yellow Collagen = blue Cytoplasm = red

Adrenal gland (x 100 oil immersion) Early fibrin deposits = diffuse yellow staining Collagen = blue Glandular tissue = red

6) Masson’s Trichrome �Trichrome stain. �Iron-haematoxylin plus two anionic dyes. �MSB is a variation of this. �Iron-haematoxylin. �Scarlet-acid fuchsine. �Light green (more of a turquoise stain).

Bladder MSB (Left), Masson’s (Right) Nuclei = black Cytoplasm including muscle and epithelium = red Erythrocytes = red Collagen = bluey green or turquoise

Tongue (low power) MSB Collagen = bluey green Cytoplasm of epithelium and skeletal muscle = red

Summary

Further Resources and OERs � Histological and Histochemical Methods. 4 th Edition. By JA Kiernan. 2007. Scion publishing. Available: http: //www. scionpublishing. com � Histopathology: Fundamentals of Biomedical Science. By G Orchard and B Nation. 2012. Oxford University Press. Available: http: //www. oup. com/uk/orc/bin/fbs/ � Laboratory skills open educational resources. De Montfort University. Available: https: //www. youtube. com/user/biologycourses