2-8. Methyl Ethyl Green -- Pyronin for Plasma Cells (Ref 4)
Staining Procedures For Plastic Embedded Tissue
Verified at the Applications Laboratory of the Biomedical Division, Sorvall
Microtomes
SOLUTIONS:
| Purification of Methyl Green |
|
|
|
Add 2g methyl green (Commission Certified only)
to 100ml hot distilled water. When cool, in separatory funnel,
extract the solution with 50ml aliquots of chloroform until
the chloroform is nearly colorless. When extraction is complete,
leave aqueous solution unstoppered overnight to allow for complete
chloroform evaporation. |
|
|
|
|
| 2% Pyronin Y |
|
2.0g |
|
Pyronin Y |
|
100.0ml |
|
Distilled water |
|
|
|
|
| 0.1 M Acetic Acid |
|
5.72ml |
|
Glacial acetic acid |
|
qs to 1000.0ml |
|
Distilled water |
|
|
|
|
| 0.1 M Sodium Acetate |
|
8.2g |
|
Sodium acetate |
|
qs to 1000.0ml |
|
Distilled water |
|
|
|
|
| Staining Solution pH 4.8 |
|
2.0ml |
|
Methyl green, 2% |
|
3.0ml |
|
Pyronin Y, 2% |
|
1.0ml |
|
Ethanol, absolute |
|
26.4ml |
|
Sodium acetate, 0.1 M |
|
17.6ml |
|
Acetic acid, 0.1 M |
STAINING PROCEDURE:
- Rinse in distilled water.
- Stain one slide at a time in methyl green -- pyronin stain for
5 minutes.
- Rinse in two changes of distilled water using 1-2 dips.
- Dehydrate in 95% ethanol for 30 seconds.
Blow dry.
Mount.
RESULTS:
Nuclei, but not most nucleoli, stain with methyl green. Most nucleoli,
cytoplasm of plasma cells, plasmacytes, plasmablasts and large pyroninophilic
cells are basophilic stem cells.
Warning: Some of the chemicals
used for the staining procedures given in this section may be hazardous
if misused. For this reason, read and observe all warnings and cautions
provided by the manufacturer for each chemical before proceeding
with a staining procedure.
Note: In order to prevent sections from
loosening from the slides during staining, all sections should
be heat-fixed (60ºC to
100ºC) to the slides for a minimum of 2-5 minutes prior to staining,
preferably at the time the sections are mounted on the slides. |
