Experimental procedure 1. Sudan III (â…£) staining method of fat:
1) Rinse frozen slices with 70% ethanol for no more than 30s.
2) Slice into Sudan III (â…£) dyeing solution for 3 ~ 15min or extend to 1h.
3) New 50% ~ 70% ethanol differentiation, until the floating color on the slice is washed away, washed with distilled water.
4) Lightly dye nuclei with alum hematoxylin diluted 1 times for 1 min or slightly longer.
5) Use filter paper to absorb the moisture from the slice and surrounding area.
6) Seal with glycerin or glycerin gelatin.
Judgment of the results: the fat is orange-yellow or orange-red and the nucleus is light blue.
2. Common staining methods for connective tissue
1) Mallory trichrome staining method: commonly used to determine the degree of disease and repair of various tissues and organs, and to distinguish the fiber components and smooth muscle in tumor tissues
Dyeing steps:
A. Fix tissue with neutral formaldehyde solution, paraffin section, and routinely dewax to water.
B. Potassium dichromate solution for 10min.
C. Rinse with distilled water for 2 minutes and distilled water twice.
D. Acid reddish solution for 2min, rinse with distilled water.
E. Aniline blue solution for 20min, 95% ethanol quickly differentiated.
F. Dehydrate directly with absolute ethanol, xylene transparent, and sealed with neutral gum.
Judgment of results: Collagen and reticular fibers are blue.
2) Van Gieson bitter acid redness method: it can show the damage, repair and sclerosis of tissues and organs, especially for the identification of collagen fibers and smooth muscle fibers in tumor tissues, which can provide an important basis for diagnosis.
Dyeing steps:
A. Tissue sections are washed with regular dewaxing and water.
B. Stain the nuclei with Weigert hematoxylin solution for 5 ~ 10 min.
C. Wash with tap water for a few minutes.
D. Check the staining degree of the cell nucleus with a microscope. Too deep can be differentiated with 0.5% hydrochloric acid ethanol.
E. Wash with tap water to blue; wash with distilled water.
F. Stain with Van Gieson solution for 1 ~ 5 min.
G. Drain the staining solution and directly differentiate and dehydrate with 95% ethanol.
H. Dehydrated with absolute ethanol, transparent xylene, and sealed with neutral gum.
Result judgment: collagen fibers are dark pink, muscle fibers, cytoplasm and red blood cells are yellow, and the nucleus is brown-black or blue-black.
3) Mesh fiber staining-Wider staining method: It can be used to display the destruction of the mesh scaffold of the diseased tissue. Especially in the pathological diagnosis of tumors, mesh fiber staining is of great value for identifying malignant tumors derived from epithelial tissues and mesenchymal tissues.
Dyeing steps:
A. Dewax the tissue section to water.
B. 10% phosphomolybdic acid, 2 ~ 5min. Rinse with distilled water for 5 minutes.
C. 1% uranium nitrate, 5s. Rinse with distilled water for 10s.
D. Silver oxide solution, 5 ~ 10min.
E. Quick wash with 95% ethanol, 1 ~ 2s.
F. Reduction with reducing solution, 1 ~ 2s. Rinse with distilled water, 2min.
G. 0.2% gold chloride color adjustment, 2 ~ 20s. Rinse with distilled water for 5 minutes.
H. 5% sodium hyposulfite, 2min. Rinse with distilled water, 2min.
I. Nuclear fast red stained cell nuclei, 5 ~ 10min. Wash with water.
J. Conventional anhydrous ethanol dehydration, xylene transparent, and neutral gum sealing.
The result is judged that the network fiber is black and the cell nucleus is red.
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