Gladstone Institutes Histology and Light Microscopy Core
Picrosirius red with fast green staining Protocol
This stain can be performed with or without the fast green for the demonstration of collagen.
Sirius red is specific for type 1 collagen in tissues, but also some type 4 and complement under
bright field. The technique is specific for collagen, however, when the dyes birefringence is
viewed under plane polarized light.
Tissue:
Formalin fixed paraffin embedded tissue sectioned at 2-10m
Controls:
collagen-containing tissues
Solutions:
0.1% Sirius red in sat sq picric acid
Stable for 3yrs and can be used many times
Sirius red F3B
0.5g
(Direct red 80, Sigma 365548)
Fast green FCF (Sigma F7258) 0.5g
Sat aq picric acid
500ml
To perform the technique without fast green then just omit this dye from the working solution
0.5% acidified water
acetic acid
tap water
5ml
1000ml
Procedure:
1. Dewax and rehydrate to water
2. Stain in Weigert’s working solution
10 min
3. Rinse gently in diH2O
10 min
4. Picrosirius red with fast green
1hr
One hour gives near equilibrium, less time stains collagen red but does not exhibit good
birefringenece
5. Acidified water
2 x rinse
6. Acidified water
1 min
7. Shake slides dry
8. 100% alcohol
2 x 1 min
9. Xylene
3 x 1 min
10.Mount in DPX (Gurr)
Results:
Collagen
Muscle / RBCs
Nuclei
Background
26 Dec 2012
- Red
- Yellow
- black
- Light green
Picrosirius red with fast green
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Under bright field collagen is red on a green (with fast green) or pale yellow (without fast
green) background. Under crossed polarized light collagen only glow bright yellow or orange,
and the thinner ones, including reticular fibres are green.
Reference:
http://www.ihcworld.com/_protocols/special_stains/sirius_red.htm
Junqueira LCU, Bignolas G, Brentani RR. Picrosirius staining plus polarization microscopy, a
specific method for collagen detection in tissue sections. Histochem J 1979; 11, 447-455
http://www.microscopyu.com/articles/polarized/polarizedintro.html
Images:
Liver section stained with picrosirius red with
fast green. The Sirius red picks up the
collagen around the vessels and hepatic
portal plates
10x
40x
26 Dec 2012
Picrosirius red with fast green
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Liver section stained with picrosirius red
without fast green, viewed under bright
field on the left panel and demonstrating
plane polarized light birefringenece on the
right
20x
26 Dec 2012
Picrosirius red with fast green
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