Atlas of plant and animal histology

Toluidin blue staining is useful to get a fast staining of animal tissues. The procedure is easy, and the staining time is very short. In addition, toludin blue provides a metachromatic staining, with colors from purple to green, through a variety of blueish

Procedure

8 µm thick sections from fixed and paraffin embedded samples will be stained. The sections are attached to gelatin coated slides.

1.- 2 x 10 min in xylene

2.- 2 x 10 min in 100º ethanol

3.- 10 min in 96º ethanol

4.- 10 min in 80º ethanol

5. 10 min in 50º ethanol

6.- 5 min in distilled H₂O

7.- 30 s in toluidine blue (C.I. 52040) 0.5 % solved in distilled H₂O with 1 % glacial acetic acid.

8.- 10 s in 96º ethanol, gentle agitation

9.- 2 x 10 s in 100º ethanol, gentle agitation

10.- 2 min + 5 min in xylene

11. Mounted with mounting media and coverslipped

Results

Nuclei: blue

Carbohydrates: blue to purple.

Cytoplasm, RNA: blue to brilliant green.

Notes

The desire staining intensity is mainly achieved during the dehydration steps, after the dye. The dips have to be quick and with gentle agitation to remove the dye excess in the tissues.

Ammonium molybdate (0.5 % in distilled H₂O) for 5 min can be added after the dye and before dehydration. Molybdate makes it more difficult to remove the dye during dehydration.

Products

Xylene

50º, 70º, 80º, 90º, 96º and 100º ethanol

Toluidine blue (C.I. 52040)

Glacial acetic acid

Ammonium molybdate

Distilled H₂O

Mounting medium

Labware

Staining dishes

Slide racks

Coverslips