[Histonet] Luxol fast blue/cresyl violet stains



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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