This staining procedure is a modification of the Mallory's trichrome. Heidenhain's azan trichrome reveals more tissular structures, particularly stains cell nuclei with an array of colors. The procedure has to be tested and adapted to each type of sample.
Procedure
Samples are fixed, embedded in paraffin, and 8 µm thick sections are obtained, which are mounted on gelatin coated slides. If the fixative contains picric acid, the deparaffined sections are incubated in 1% of aniline in 96º ethanol for 30 min.
1.- Xylene, 2x10 min.
2.- 100º ethanol, 2x10 min.
3.- 96ºethanol, 10 min.
4.- 80º ethanol, 10 min.
5.- 50º ethanol, 10 min.
6.- Distilled H2O, 5 min.
7.- Azocarmine, previously heated at 60 ºC, for 1 h at 60 ºC.
Azocarmine G
0.1 g azocarmine G (C.I. 50085)
200 ml distilled H2O
2 ml acetic acid
8.- Rinse several ties in distilled H2O.
9.- Differentiation by several dips in ethanol-aniline (1 % of aniline (C.I. 76000) in 96º ethanol).
The differentiation speed depends on the proportion of aniline and alcohol concentration. Thus, to get different results, the aniline-ethanol ratio can go from 1 % aniline - 70º ethanol to 0.1 % aniline - 96º ethanol.
10.- Stop the differentiation with acetic acid ethanol (1 % acetic acid in 96º ethanol).
11.- Rinses in distilled H2O.
12.- 5 % phosphotungstic acid, 1 h.
Phosphotungstic acid is a mordant that favors the staining. At the same time, it differentiates the azocarmine staining.
13.- Several rinses in distilled H2O.
14.- Heidenhain's blue, 2 h.
Heidenhain's blue
0.2 g aniline blue (C.I. 42755)
0.5 g orange G (C.I. 16230)
100 ml distilled H2O
1 ml acetic acid
15.- 96º ethanol, a fast dip.
16.- 100º ethanol, several fast dips.
17.- Xylene, 2x10 min.
Results
Nuclei: different colors, from orange to red
Cytoplasm: several colors
Muscle: from orange to red
Collagen: blue
Notes
The procedure may be modified according to the sample features.
Differentiation steps are critical for the staining quality.
The final dehydration has to be very fast, otherwise the blue color fade away.
Products
Xylene
50º, 80º, 90º, 96º y 100º ethanol
Azocarmine G (C.I. 50085)
Aniline (C.I. 76000)
Aniline blue (C.I. 42755)
Orange G (C.I. 16230)
Phosphotungstic acid
Acetic acid
Distilled water
Mounting medium
Labware
Staining dishes
Stove at 60 ºC
Slide grid
Coverslip
