[Histonet] Bodian Stain

Thu Jan 8 09:26:52 CST 2004

How about calibrating a waterbath to 37C and using that, avoiding a
convectional drying oven.  Once the waterbath is calibrated, then you know
settings for next go 'round of Bodian.  Waterbath heating is superior to
drying ovens as the heating is consistently even.  

At 05:42 AM 1/8/2004 -0500, you wrote:
>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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>> Histonet <@t> lists.utsouthwestern.edu
>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>>
>
>
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>
>
Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology 
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)