In animal tissues, dyes are important to distinguish the nucleus from the cytoplasm and from the extracellular matrix. In plant tissues, dyes stains the cell walls, as well as cell components. A common dye in plant histology is safranin, used to for staining lignified cell walls (secondary walls) and Alcian blue (or fast green) to stain non-lignified cell walls (primary cell walls).
Procedure
Samples fixed with FAA (formaldehyde, alcohol and acetic acid) are processed. Sections can be obtained with a vibratome, freezing microtome or paraffin microtome. Thick sections are handled with small brushes and in small wells. Paraffin sections are attached to gelatin coated slides and processed in staining dishes. Different incubation times are set depending on the type of sections. The following procedure is for paraffin sections, so that paraffin is removed with xylen and sections are hydrated in a descending ethanol gradation.
Safranin / Alcian blue
1.- 50o ethanol, 5 min.
2.- Safranin. 1 min.
Safranin (C. I. 75100): 1 g
95o ethanol: 15.5 ml
Distilled H2O : 14.5 ml
Before use, mix the solution (stock solution) with 50o ethanol (1:1)
3.- Distilled H2O, 4 x 20 s
4.- Alcian blue (1 %, pH 2.5, 3-5 min
Alcian blue (C.I. 74240): 1 g
Distilled H2O: 97 ml
Glacial acetic acid: 3 ml
Agitate for 1 h and filter. The solution can be used for several years.
5.- Distilled H2O destilada, 1 min
6.- Differentiation with 96o ethanol until the section is stained in pale blue. The staining process can be checked under the binocular microscope.
7.- 100o ethanol, 1 min
8.- Xylene, 1 min
9.- Mounting medium and coverslipped
Safranin / fast green
1.- 50o ethanol, 5 min
2.- Safranin (1 %, pH 2.5), 3 - 5 min
Safranin (C. I. 75100): 1 g
95o ethanol: 15.5ml
Distilled H2O: 14.5 ml
Before use, mix the solution (stock solution) with 50o ethanol (1:1).
3.- Distilled water H2O, 4 x 20 s
4.- 96o ethanol, 1min
5.- Fast green (1%), 60s.
This dye competes with safranin, so that much green color means less red (safranin) staining.
100o ethanol: 50 ml
Clove oil: 50 ml
Fast green (C. I. 42053): 1 g
6.- 100o, ethanol 1min
7.- Xylene, 1 min
8.- Mounting medium and coverslipped
Resultados
Primary cell walls: blue (Alcian blue), or green (fast green).
Secondary cell walls: red.
Cell walls with cutine or suberin: red.
Notes
In general, a quick staining procedure means the use of vibratome or freezing microtome sections. However, good staining in good quality tissues can be obtained in 5 to 10 µm thick paraffin sections.
Products
Xylene
50º, 70º, 90º, 100º ethanol
Glacial acetic acid
Clove oil
Safranin (C. I. 75100)
Alcian blue (C.I. 74240)
Fast green (C. I. 42053)
Distilled H2O
Mounting medium
Labware
Plate with small wells
Brushes
Slides
Coverslips
Magnetic stirrer
