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

2.  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.

3.  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.

5.  Tolonium Chloride  Useful blue cationic dye  Cheap and simple application

9.  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.

11.  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?

12. Liver tissue with no counterstain Reticulin = black

13. Liver counterstained with what? Reticulin = black Cytoplasm = pink

14.  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.

15. Bladder Collagen = red All other tissue including transitional epithelium = yellow

16. 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.

17.  Trichrome stain.  Martius yellow and phosphotungstic acid.  Brilliant crystal scarlet.  Methyl blue.  What do the dyes stain?

18. Epithelium = red Collagen = blue Cytoplasm = red No visible yellow

19. Collagen = blue Erythrocytes and early fibrin = yellow Cytoplasm = pink

20. Erythrocytes clearly yellow Collagen = blue Cytoplasm = red

21. Early fibrin deposits = diffuse yellow staining Collagen = blue Glandular tissue = red

22.  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).

23. Nuclei = black Cytoplasm including muscle and epithelium = red Erythrocytes = red Collagen = bluey green or turquoise

24. MSB Collagen = bluey green Cytoplasm of epithelium and skeletal muscle = red

26.  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