Sirius red is a strong acidic anion dye, which is easy to react with the basic group in collagen fiber, enchancing birefingence and improve resolution. It is closely adsorbed with collagen and is extremely stable. Therefore, the stain is not discoloured and is specific, which is the best dye for collagen staining. Under polarized light microscope, different types of collagen display different solors and contrast bright.
1.Deparaffinage and hydrate to water: following next steps, put paraffin sliced into XyleneⅠ20 mins,and XyleneⅡ 20 mins, anhydrous ethanol 5mins, and 75% anhydrous ethanol 5 mins, at last tap water is washed.
2.The sirius Red Stain：the slices were cut into Sirius red stain solution 8-10 mins, two or three tanks dehydrated with anhydrous ethanol.
3.Dehydrate seal: Slice and put in clean xylene transparent 5 mins,Glycerol Jelly seal.
4.Microscopic examination and image collection analysis.
Under normal light microscope, the collagen fibers were red and the others were yellowish. Under polarized light microscopy, the I-type fiber are closely aligned, showing a strong birefringence, with yellow or red fibers. And Ⅱ-type fiber, showing weak double refraction, a greenish fine fiber. Ⅳ type fiber, showing weak double refraction basilemma, pale yellow.
1．Observe collagen fiber birefraction immediately after staining. Otherwise, the birefraction may fade or even disappear, which may cause the false positive result.
2．Strictly manage the dehydrate time to remove residual staining solution but not over-dehydrate to affect the yellow background.
3．The time can be extended to 60 mins for tissue infiltration of the collagen with less collagen and the color of collagen is more vivid.