[Histonet] Bodian Stain

Thu Jan 8 04:42:21 CST 2004

I don't think Bodian would work in a microwave, as copper shot is used in
the coplin jar. That's a metal, and I think it could cause arcing.

I've done the Bodian, leaving it incubate overnight, such as putting it in
at 4 pm and taking it out at 7-8 am. It works.

Is there a way they could turn their drying oven down to the 37 degree C
just for overnight? Or over the weekend?

Below is our procedure.

Peggy A. Wenk, HTL(ASCP)SLS
William Beaumont Hospital
Royal Oak, MI 48073

NERVE FIBERS - BODIAN

PREPARED BY: Peggy A. Wenk, BS, HTL(ASCP)SLS

ADOPTED BY: ____________________________________
DATE:___________________________

 ADOPTED
REVIEWED
REVIEWED
REVIEWED

SUPERSEDES:

PURPOSE:
This stain demonstrates nerve fibers, nerve endings, and neurofibrils, which
are aggregates of microtubules and neurofilaments found in the cell body,
axon and dendrites of nerve cells.

PRINCIPLE:
The reaction is an argyrophilic silver stain. Protargol, a silver proteinate
compound, impregnates the tissue sections. Copper, which is added to the
incubating solution, is more reactive than the silver, and will remove the
silver from the connective tissue. This allows for a greater differentiation
between the neural fibers and the connective tissue. Hydroquinone and
formaldehyde reduce the silver salts to visible metallic silver. Gold
chloride tones the section. Oxalic acid is used to reduce the gold. This
gives a darker stain, as the metallic gold is also deposited onto the
tissue. Sodium thiosulfate removes excess unreduced silver.

FIXATION:
Any well fixed tissue.
10% neutral buffered formalin preferred.
Avoid mercuric fixatives.

TECHNIQUE:
Cut routine paraffin sections at 8-10 um.

CONTROL:
Section of brain medulla or peripheral nerve.

QUALITY CONTROL:
1. This is a capricious stain. Follow procedure exactly. Do NOT try to
reduce percentages or time.
2. The quality of the silver proteinate seems to be critical to the success
of this stain.

EQUIPMENT:
Balance, Erlenmeyer flasks, graduated cylinders, acid clean coplin jars,
non-metal forceps, 37o C. oven.

 CAUTION:
Follow standard safety procedures when preparing stains.

SILVER PROTEINATE (PROTARGOL) is an irritant and is possibly toxic.
HYDROQUINONE is an irritant to eyes, skin and respiratory system.
FORMALDEHYDE is a poison. May be fatal or cause blindness if swallowed.
Cannot be made
  non-poisonous. Possible cancer hazard. Irritating to eyes, skin and
respiratory tract.
 Can cause severe eye burns.
GOLD CHLORIDE is an irritant to eyes and skin.
OXALIC ACID is a strong reducing agent. Contact with other material may
cause fire. May
  cause skin and eye burns. Irritating to respiratory system.
SODIUM THIOSULFATE is an irritant.

REAGENTS:
PROTARGOL (SILVER PROTEINATE) SOLUTION
Protargol (silver proteinate)  0.5 g
Distilled water  50.0 mL
Place the distilled water in a 50 mL beaker. Sprinkle Protargol on the
surface of the water. Do NOT shake or stir. Place in 37o C. oven for about
30 minutes, until it is dissolved. Make solution just before use. Discard
after use.

REDUCING SOLUTION
Hydroquinone (C6H4(OH)2)  1.0 g
Distilled water   50.0 mL
Formaldehyde, 37-40% (HCHO)   2.5 mL
JUST BEFORE USE, dissolve together hydroquinone in distilled water. Add
formaldehyde. Discard after use.

1% GOLD CHLORIDE
Gold chloride (HAuCl4C3H2O)  1.0 g
Distilled water  100.0 mL
Dissolve together. Store at room temperature. Stable for months. May be
reused. Filter when necessary.

2% OXALIC ACID
Oxalic acid ((COOH)2C2H2O)  2.0 g
Distilled water  100.0 mL
Dissolve together. Store at room temperature. Stable for months.

5% SODIUM THIOSULFATE
Sodium thiosulfate (Na2S2O3)  5.0 g
Distilled water  100.0 mL
Dissolve together. Store at room temperature. Stable for months.

PROCEDURE - Bodian:
1. Deparaffinize and hydrate slides through graded alcohol to distilled
water.

2. Place 3 g copper shot in an acid-clean coplin jar.

3.  Pour protargol solution over copper shots.

4. Place slides in protargol solution.

5. Incubate slides in 37o C. oven 24-72 hours

6. Rinse in 3 changes of distilled water 5 seconds each

7. Place in reducing solution  10 minutes

8. Rinse in distilled water, 3 changes 5-10 seconds each

9. Tone in 1% gold chloride 5 minutes

10.  Rinse in distilled water, 3 changes 5-10 seconds each

11. Develop in 2% oxalic acid until background is gray
and nerve fibers appear clearly black 3-5 minutes

12. Rinse in distilled water, 3 changes 5-10 seconds each

13. Place in 5% sodium thiosulfate 5 minutes

14.  Wash in running water 10 minutes

15.  Dehydrate through graded alcohols and clear in xylene.

16.  Coverslip with a synthetic mounting media.

RESULTS:
Nerve fibers - black
Connective tissue - gray to black
Background - gray/purple

PROCEDURAL NOTES:
1. If copper shots appear "rusty", clean them by placing in a solution of
aqua regia (15 mL hydrochloric acid, concentrated, and 5 mL nitric acid,
concentrated). Use gloves and apron when preparing or handling this
solution. Prepare and use under hood. Wash copper shots in running water,
and then distilled water, before using in protargol solution.

2. Leave the Protargol to dissolve from the surface downward. Do not disturb
until dissolved. Do not allow to coagulate.

3. Prolonged treatment in oxalic acid will destroy the protargol reaction.

4. Use acid-cleaned coplin jars and non-metal forceps, or a dirty background
may appear.

5. Nuclear fast red may be used as a counterstain. Nuclei will be stained
pink.

6. Use 1% gold chloride, not the more dilute used for most silver stain
toning. Tone for entire 5 minutes. Using lower percentage gold chloride or
less time will cause the nerves and background not to turn to the
characteristic Bodian purple-black.

REFERENCES:

Bancroft JD, Stevens A: Theory and Practice of Histological Techniques, 3rd
ed. New York, NY, Churchill Livingstone, 1990.

Carson FL: Histotechnology: A Self-Instructional Text, Chicago, IL, ASCP
Press, 1990.

Sheehan DC, Hrapchak BB: Theory and Practice of Histotechnology, 2nd
edition. Columbus, Ohio, Battelle Press, 1980.

Vacca LL: Laboratory Manual of Histochemistry, New York, New York, Raven
Press, 1985.

* Actual source of procedure is unknown.

----- Original Message -----
From: "Atoska S. Gentry" <gentras <@t> vetmed.auburn.edu>
To: "Histonet" <histonet <@t> pathology.swmed.edu>
Sent: Tuesday, January 06, 2004 3:42 PM
Subject: [Histonet] Bodian Stain

> Hello, a former colleague of mine who works in the only local human
> pathology lab is seeking input on the availability of a protocol for
Bodian
> Stain other than the one in the AFIP manual. The reason they are seeking
> this is their lab is only equipped with one incubator and they routinely
> use it for drying slides at temperatures much higher than the required
> 48hour, 37C incubation of the protargol. They are equipped with a
microwave
> for staining but are not sure how to use it for drying of slides without
> trial and error. Any suggestions provided will be much appreciated.
Thanks,
> Atoska p.s. our lab is equipped but we only process animal tissues here.
> We've provided them with the chemicals they were lacking to complete this
> stain.
>
> Atoska S. Gentry B.S., HT(ASCP)
> Research Assistant III
> Scott-Ritchey Research Center
> College of Veterinary Medicine
> Auburn University, AL  36849
> Phone# (334)844-5579  Fax# (334)844-5850
>
>
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