Re: [Histonet] Luxol fast blue/cresyl violet stains

From:Rene J Buesa

  When you say "5 slides from the same animal" do you mean slides from different blocks from the same animal or slides from the same block.
  If the slides are from different blocks then the problem could reside in the blocks and how they were treated. If all the slides are from the same block then the difference could reside in the configuration the slides were placed in the staining jar (although this explanation could be far fetched).
  I don't think that the differences could be due to the differentiation step because you say that after differentiation the slides are placed in distilled water, had they been "waiting" for the result of one slide to stop the differentiation on the others that could be the cause.
  Now, if the 37Celsius incubation is done in an oven and the oven does not have a good air circulation it could be that one area in the staining jar could have a difference in temperature at least during some time, although that could be compensated during the long incubation period. So I really don't know what to atribute the problem to.
  Not exactly these, but the delay itself, prompted us to eliminate this protocol and go to one using microwaves (MW) as follows:
  1- dewax and take sections to absolute ethanol (EthOL)
  2-place slides in plastic Coplin jar and add 95% EThOL
  3- heat slides in the 95% EthOL for 15 secs. at 0.95 kcal ("Level 5" of our 480 W MW oven which was equivalent to 266 Watts)
  4- dump the heated 95% EthOL (used only to heat the slides) and substitute it with 40 mL of Luxol Fast Blue (LFB). Heat at the same level (266 Watts) for 20 secs (=1.27 kcal).
  5- transfer the Coplin jar with the slides to a water bath at 60Celsius during 5 minutes.
  6- Wash slides in distilled water.
  7- Differentiate the slides , one at a time, with 0.5%aq. lithium carbonate solution until no more blue color runs off the slides (not necessary under the microscope)
  8- Transfer the slides to distilled water.
  9- Dump the dist. water and add the Cresyl Echt Violet (CEV) and heat in the MW at full power (480 Watts) for 20 secs.(=2.3 kcal).
  10-Transfer the slides to the water bath at 60Celsius for 5 minutes.
  11- Dehydrate QUICKLY, clear and mount as usual.
  With this procedure we did not have any problems and it could be completed in about 30 minutes. Perhaps you could care to try this protocol to find out if the problem persists.
  This is how we did it in our lab. Hope this will help!
  Rene J. wrote:

I have a question concerning the lfb/cv stain. This is the situation we have in our lab. The tissue we are working on is Bouin's fixed mouse brain and spinal cord (cord is left in the vertebrae). The spinal cord may be left in Bouin's for an extended period for decalcification (two-three weeks). The trimmed tissue is then sent to our lab where we rinse it for a day before processing on our VIP processors. The next day we embed the tissue and then it is cut by one of our technicians and stained. This is our staining technique: deparaffinize to 95% alcohol and place in alcoholic luxol fast blue overnight at 37 degrees. The next morning it is rinsed twice in 95% alcohol and twice in distilled water. The slides are separated into racks containing either brain or spinal cord for differentiation. Slides are dipped in 70% alcohol for 20 seconds then lithium carbonate for 20 seconds. This is followed by two rinses in distilled water. Then checked microscopically before determining if
 differentiation is complete or not. If not, then the slides go another round through 70% and lithium for a few more seconds. Once this part is done the slides are put into a warmed cresyl violet solution (we add 10% glacial acetic acid, 10 drops for every 100mls of stain) for 4-6 minutes then rinsed in 95% alcohol three times. Finally they are dehydrated, cleared and coverslipped.

The problem comes when we have 5 slides from the same animal, all processsed and stained together, where in one slide the cresyl violet works but in the other 4 it doesnt' work. Every slide is treated exactly the same, yet we have this difference. Any ideas on what may be happening? We've discussed decalcification and possible lithium effects on the cresyl violet. We're at a loss and our pathologist is very determined to discover the solution for this problem.

Thank you advance for any ideas you may send our way..........We greatly appreciate your help!

Judi Ford
HIstopathology & Microscopy
The Jackson Laboratory
600 Main St.
Bar Harbor, Me 04609

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