This Masson Trichrome Staining is modified from Mallory trichrome staining. The differential visualization of tissue elements is achieved with two or three similar anionic dyes. A dye with a small molecular size is used first which stains all tissue elements in the section. The color stain is selectively removed from unwanted areas, which also acts as a mordant for the next staining step. A dye with a large molecular is then progressively applied to the section. After the staining, tissue shows multiple colors in different elements. The coloring principle is based on the different size of dye molecular and tissue permeability.
Potassium dichromate mordant
Weigert hematoxylin iron solution A
Weigert hematoxylin iron solution B
Acid ponceau fuchsin solution
Phosphomolybdic acid solution
Aniline blue solution
1 Deparaffinize and rehydrate by immersing the slides through the following:
a. Xylene: two washes 20 minutes each
b. 100% alcohol: two washes for 5 minutes
c. 75% alcohol: wash for 5 minutes
d. Wash briefly in tap water
2Potassium dichromate stain
Rinse slides in Potassium dichromate mordant solution overnight, wash in tap water quickly.
3 Iron hematoxylin stain
Mix up theWeigert hematoxylin iron solution A with solution B by equivalent volume to generate iron hematoxylin solution. Rinse the slides in this iron hematoxylin solution for 3 minutes, wash in tap water. Then differentiate in 1% acid alcohol for 30 seconds, wash in running tap water.
4 Acid ponceau fuchsin stain
Stain in acid ponceau fuchsin solution for 3-5 minutes, rinse in tap water to wash.
5 Phosphomolybdic acid stain
Immerse slides in phosphomolybdic acid solution for 1-3 minutes. Do not wash in water.
6 Aniline blue stain
Stain the slides with aniline blue solution for 3-6 minutes.
Differentiate slides in 1% glacial acetic acid solution, dehydrate in 3 changes of absolute alcohol, 5 minutes each.
8 Clearing and mounting
Clear in xylene for 5 minutes, mount with resin mounting medium.
9 Microscopy examination and analysis
Collagen fiber should be blue; muscle fiber, cellulose and red blood cells should be red.
1 The mixture of Weigert hematoxylin iron solution A and B should be prepared when it is needed. The mixture is inclined to be oxidized and do not work.
2 After stain in potassium dichromate, slides should be washed in tap water to remove residual dye as quickly as possible in a short time.
3 The iron hematoxylin mixture can be repeatedly used for staining but should be changed according to the slides.
4 Ensure the proper staining extent by ponceau. If the red color of collagen is too deep, it will adversely affect the aniline blue staining.
5 In the step 7, manage the proper differentiate. The over-differentiate would weaken the blue color, and no enough differentiate may cause the colors’ overlay of blue and red.
The heart tissue stained by masson stain. The collagen fiber shows blue, muscle fiber and red blood cells show red.
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