Picrosirius red method1. Used to stain collagen I and III. The stain will quantify the amount of collagen in a given area of myocardial tissue, i.e. the collagen area fraction. Picrosirius Red Stain binds specifically to collagen fibrils of varying diameter that is used to distinguish collagen type I from collagen type III. Collagenous structures of the mandible stain brilliant red. Dentinal tubules, Sharpey's fibers and other structures not easily seen in sections stained with hematoxylin and eosin alone can be seen clearly after this procedure. Under polarized light collagen fibers can be specifically identified and their orientation determined. Picrosirius red-hematoxylin is recommended for examination of normal or pathologic dental specimens.
Contents of Kit:
- Solution A
- Solution B
- Solution C
Fixation:
Fixation is not critical. The method is most frequently used on paraffin sections of objects fixed adequately (at least 24 hours but ideally 1 or 2 weeks) in a neutral buffered fomaldehyde solution.
Procedure:
1. Deparaffinize and hydrate to distilled water.
2. Stain in Weigerts Hematoxylin for 8 minutes (optional) if Weigert's hematoxylin is not used, go directly to step four.
3. Rinse well in distilled water.
4. Place in Solution A for 2 minutes.
5. Distilled water rinse.
6. Place in Solution B for 110 minutes.
7. Place in Solution C for 2 minutes.
8. 70% Ethanol for 45 seconds.
9. Dehydrate, clear and mount.
Results:
Stains fibrillar type I and type III collagen.
- Collagen = Red
- Type I = Yellow
- Type III = Green
References:
Puchtler H, Waldrop FS, Valentine LS. Polarization microscopic studies of connective tissue stained with picro-sirius red FBA. Beitr Path 1973; 150, 174-187
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
Whittaker P. Polarized light microscopy in biomedical research. Microscopy and Analysis 1995; 44, 15-17
Whittaker P, Kloner RA, Boughner DR, Pickering JG. Quantitative assessment of myocardial collagen with picrosirius red staining and circularly polarized light. Basic Research in Cardiology 1994; 89, 397-410