http://www-medlib.med.utah.edu/WebPath/HISTHTML/MANUALS/HOLZER.PDF

PURPOSE: An appropriate term for glial fibers is 'nerve glue', because
they provide the internal support of the central nervous system. There are
four types of glial cells: astrocytes, oligosendroglia,microglia, and
ependymal cells.
PRINCIPLE: The glia fibers are stained with crystal violet which are
resistant to the aniline-chloroform differentiating solution.
CONTROL: Cerebral cortex
FIXATIVE: 10% formalin
TECHNIQUE: Cut paraffin sections at 6μ.
EQUIPMENT: Rinse all glassware in DI water, coplin jars, bibulous paper.
REAGENTS:
Phosphomolybdic Alcohol:
0.5% phosphomolybdic
acid 20.0 ml
95% alcohol 40.0 ml
Make fresh, discard after use.
CAUTION: flammable, avoid contact and
inhalation.
Absolute Alcohol-Chloroform:
Absolute alcohol 40.0 ml
Chloroform 160.0 ml
Make fresh, discard after use.
CAUTION: Carcinogen, flammable.
10% Potassium Bromide:
Potassium bromide 10.0 gm
Mix well. Solution is stable for 3
months.
Holzers Crystal Violet:
Crystal violet 5.0 gm
Absolute alcohol 20.0 ml
Chloroform 80.0 ml
Mix well. Solution is stable for 3
months.
CAUTION: Carcinogen, flammable.
CAUTION: Avoid contact and inhalation.
Differentiating Solution:
Aniline oil 30.0 ml
Chloroform 45.0 ml
Ammonium hydroxide 5 drops
Make fresh, discard after use.
CAUTION: Carcinogen, flammable,
corrosive.
NERVE TISSUE
HOLZER'S Page: 2 of 3
SAFETY: Work in fume hood, wear nitrile gloves, goggles and lab coat.
Avoid contact and inhalation.
Phosphomolydic acid; irritant to skin and eyes.
Chloroform; carcinogen. Toxic on ingestion and inhalation. Target organ
effects on liver, reproductive system, fetal tissue, CNS, blood and GI
systems. Overexposure to vapor can cause unconsciousness and death.
Crystal violet (Gentian violet); carcinogen.
Aniline; possible carcinogen, neoplastic. Moderatre skin, severe eye
irritant. Sensitizer. Toxic by skin absorption. Excessive exposure may
cause blue discoloration of extremities, nusia, headache and drowsiness.
PROCEDURE:
1. Deparaffinize and hydrate to 95% alcohol.
2. Phosphomolybdic acid solution, 3 minutes.
3. Drain off solution.
4. Alcohol-chloroform solution until sections are translucent, drain.
5. Crystal violet stain, 30 seconds.
6. Drain off solution.
7. Potassium bromide solution, 5 minutes.
8. Wash in distilled water, blot dry with bibulous paper.
9. Differentiating solution, check microscopically.
10. Clear in xylene, coverslip.
RESULTS:
Glia fibers deep violet-blue
Background pale violet
NERVE TISSUE