[Histonet] FURTHER INFORMATION REGARDING THE PICROSÍRIUS-POLARIZATION METHOD
Luiz Carlos Junqueira
lcjunca <@t> gmail.com
Fri Mar 10 11:55:10 CST 2006
FURTHER INFORMATION REGARDING THE
As I have found in Internet questions regarding the use of the picrosírius-polarization method, these additional informations regarding its use may be useful.
1-This method considered to be "one of the best understood techniques of collagen histochemistry" presentes the following characteristics:
2-The picrosírius solution can last for more than 10 years in a dark bottle. Slides stained by it do not fade in 20 years;
3-I always pre-stain slides of paraffin embedded material initially with PS and after brief washing with Harris Hematoxylin. I feel that this produces far better results;
4-If you use cheap photographic polaroid filters you will obtain a nice blue background, but if you use them repeatedly, for long periods, you might end up with an inflammation of the conjunctiva. With more expensive microscopic polarizing filters (I use a Nikon) you will get a totally black background and with a much higher resolution;
5-This will permit you to observe birefringence in basement membranes. See Junqueira et.al-Evidence for collagen molecular orientation in basement membranes-Histochem. J.15: 785,1983;
6-I tried several ways of staining resin (historesin) embedded material with no success, probably due to Sírius high molecular weight (+-1000);
7-Picrosírius stains cartilages collagen well only after pre-digestion with papain, that extracts a cloud of proteoglycans. The figures thus obtained, remember the Saint Chapel glass windows in a sunny day;
8-Thin birrefringent collagen fibers can be adult reticular fibers (type III) or young collagen (type I) fibers. Thus, distinction between these two types of collagen fibers cannot be made during developing processes;
9-Not all Sírius Red batches work well. The only I use is the F3B200 from Mowbay Chemical Co, Union New Jersey USA. Before its use in Histology, its cost was approximately U$30,00 per pound.
10-Compared with Picrosírius, the trichrome staining procedures tested, do not stain collagen specifically or electively;
11-Picrosírius stain collagen specifically. The only exceptions are some rare secretory salivary gland cells that contain basic cytoplasmic proteins, however they are not birrefringent. In some species the queratin present in the queratinized layer of the skin can light up. No other exceptions were observed from fishes to mammals. To distinguish birrefringent from refringent material it is necessary to rotate the specimen using a microscope equipped with circularly motion. Only birrefringent material changes its color;
12-Both formaldeyde and Bouin give good results with picrosírius. Very thin sections do not give good information regarding the dispersed disposition of collagen in these sections;
13-The Picrosírius-polarization method has been widely used in research to study collagen as shown by the 57.000 references in "Sírius collagen" in Google in 06/3/2006. Figures obtained with this procedure are of exceptionally high didactic quality, and would, with great advantage, substitute the usual haematoxylin-eosin or trichrome preparations. Despite this, figures using this method described approximately 35 years ago, appeared only in 35 micrographs in a recent text book (Basic Histology, Junqueira & Carneiro, 11th edition, McGraw-Hill). More information on this method is obtainable in the review that presents most of the literature regarding its physico-chemical basis and its use in other fields (Montes and Junqueira: The use of picrosírius polarization method for the study of biopatology of collagen. Mem. Inst. Oswaldo Cruz Vol. 86, Suppl.III, 1-11,1991).
For futher information my address is:
Poços de Caldas-MG- Brazil
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