[Histonet] Bodian Stain

Thu Jan 8 09:05:13 CST 2004

Hi HistoNetters

Actually, there is a very good microwave Bodian stain.  It is 
procedure 15.23 in the new edition of Kok & Boon's book (pp 219-220). 
The staining time is reduced to 2-1/2 hours.  There will be no arcing 
if no metallic accessories are used.

best regards,
Steven Slap
Microwave Consultant

At 5:42 AM -0500 1/8/04, Lee & Peggy Wenk 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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>>
>
>
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