[Histonet] picro sirius red staining

[Galina Deyneko]

Please see my protocol, we successfully use it on murine liver and hearts.I do not stain nuclei, in my opinion counterstaining with any type of hematoxylin contaminates and fades picris acid.


 
-         Sirius Red F3BA(Polysciences #09400), (Direct Red 80 from Sigma Chem. Co. (Cat# D 030))

-         Saturated aqueouspicric acid solution (LabChem Inc. #LC 18670-2)

-         Picric acidpowder (Spectrum #P1145)

-         Phosphomolybdicacid (Sigma #HT153)

-         Hydrochloric acid(Sigma #H1758)

-     Bouin’solution

0.1% solution of Sirius Red:

0.2 gSirius Red powder

200 mlsaturated aqueous solution of picric acid

-         Add alittle amount of solid picric acid to ensure saturation. Stir well, filterbefore use. 

-         Stablefor 1 year and can be used many times.

 

02% solution of phosphomolibdic acid:

4 ml of10% phosphomolibdic acid

200 ml DIwater

 

0.01N solution of hydrochloric acid:

166 µl of38% HCL /830 µl

200 ml DIwater      /1 L

Procedure:

·        De-waxSlides

·        Incubatein 0.2% solution of phosphomolibdic acid (optional)

·        Mordantovernight in saturated solution of picric acid at room T

·        Enhancethe yellow color by mordant in saturated solution of picric acid additional 1- 2hour at 60 C in the oven.

·        Stain in0.1% solution of Sirius Red for 2 hours at RT

·        Rinse in onechanges of 0.001 N solution HCl- very quick dip!!

·        Dehydratein four  changes of  fresh!! 100% Ethanol also quick deep in each

·        Clearslide through three changes of xylene

·        Coverslipusing a permanent mounting media

Results:

The PSRed method stains mostly collagen types I, II, and III.
Light microscopy: collagenous fibers – red, other tissue elements - bright yellowPolarized microscopy:  collagen fibers - orange/red bands against black background
 
Galina Deyneko Novartis, CVM
galinadeyneko at yahoo.com