[Histonet] wrinkling of rat brain tissue on subbed slides

From:Neil Fournier

I have been having difficulty lately with rat brain tissue (40 to 50 micron thick) mounted on gelatin coated slides.  We have noticed that several sections will often show wrinkling and folds along the sides of the cortex.   In some cases, the tissue has severe folds and wrinkling throughout making them unusable for quantification.  We believe the problem emerges during our cresyl violet staining, but we have made up new solution and still have the same problems.  The sections are not falling off but they are typically wrinkled and folded along the sides; however, I must mention that it isn't every section on every slide showing the problem.    The folding is extremely annoying since we are interested in quantifying amygdaloid regions and piriform cortex

We originally thought that perhaps the slides were too old (they were subbed in July 2004); however, we have purchased new slides and have the same problem.  We have never stored our slides in the fridge, but I am thinking that we should be begin to adopt this protocol (Is there a specific temperature that the slides should be kept at?  And does one mount tissue on slides just taken out of a fridge or should the slides equilibrate at room temperature before mounting?).  

Our basic protocol is the following:  mount sections on subbed  slides (We use 0.5% gelatin and 0.05% chromium potassium sulphate) and let dry minimal 24 hrs before cresyl violet staining.   We simply place the slides on a tray lying flat with a paper towel placed on top.  After 24 hrs, we then stain the slides with cresyl violet.  Our cresyl violet protocol is as follows:  1) distilled H2O (for 1 min), 2) 100% EtOH (3 min), 3) 95% EtOH (3 min), 4 70% ETOH (3 min), 5) distilled H2O (3 min), 6) 0.1% cresyl violet (5 - 10 min) made from 1% cresyl violet stock solution, 7) distilled H20 (rinse), 8) distilled H2O (rinse), 9) 3% glacial acetic acid (4 quick dips), 10) distilled H2O (rinse), 11) 70% ETOH (1 min), 12) 95% ETOH (1 min), 13)  100% ETOH (1 min), 14) xylene (at least 5 min).   (Perhaps there is something wrong with this protocol or steps.  Any potential suggestions are welcomed).

Has anyone ever encountered this problem before and have potential solutions.  

Any help is appreciated,

Neil Fournier
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