Dr Vivien Rolfe
De Montfort University
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Educational Resource
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 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.
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.
 Tolonium
Chloride
 Useful blue cationic dye
 Cheap and simple application
 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.
 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?
Liver tissue with no counterstain
Reticulin = black
Liver counterstained with what?
Reticulin = black
Cytoplasm = pink
 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.
Bladder
Collagen = red
All other tissue including transitional epithelium = yellow
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.
 Trichrome
stain.
 Martius yellow and phosphotungstic acid.
 Brilliant crystal scarlet.
 Methyl blue.
 What do the dyes stain?
Epithelium = red
Collagen = blue
Cytoplasm = red
No visible yellow
Collagen = blue
Erythrocytes and early fibrin = yellow
Cytoplasm = pink
Erythrocytes clearly yellow
Collagen = blue
Cytoplasm = red
Early fibrin deposits = diffuse yellow staining
Collagen = blue
Glandular tissue = red
 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).
Nuclei = black
Cytoplasm including muscle and epithelium = red
Erythrocytes = red
Collagen = bluey green or turquoise
MSB
Collagen = bluey green
Cytoplasm of epithelium and skeletal muscle = red



Histological and Histochemical Methods. 4th
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