I am using FD Rapid Golgistain kit to stain and study dendritic morphology. I section the brains after impregnation and mount them on to gelatin coated slides and let them overnight at room temperature to dry. Following day I tried to wash, according to the protocol provided, but I lose a lot of sections which detach from the slides into water. I figured out that this is due to incomplete drying so I decided to let them dry for two nights but now, though the retention of sections onto slides has considerably improved, I see extracellular crystal formation that make studying spines impossible. The crystals are seen only in the low half of the sections so it has to be due to over drying. Please advice me to keep my sections on slides while washing. I am now considering exploring other methods to study dendritic morphology. Does the method of staining with Dii crystals take a long time to set up?
Also how can I distinguish cortical layers in Golgi stained brain sections?
I had this problem when I did not fix the sections on the slides. Have you fixed (on-slide) with paraformaldehyde / formalin? Personnally I would not leave the tissues to dry for that long; you could have protein degradation. Another thing that helps is to let the slides dry overnight in a vacuum chamber / dessicator.
Thank you very much Jean and Emma. Your advice will save me a lot of time and effort.
I realized that my protocol for preparation of gelatinized slides has 0.5% gelatin solution with 5 dips in contrast to the 3% gelatin solution and 10min immersion that you suggest. I will also use a warm plate to dry my sections this time. I do not have a dessicator in our lab.
I also had precipitate problem in the CA1 region and as you suggest may be the saline perfusion can help resolve this.
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