I am trying to make histological rat brain slides, pathology is doing embedding, and I am slicing the blocks on microtome, thickness 1,5 micrometers. Procedure is slicing, cold water, warm water, putting it on slides. The problem is when the tissue comes to warm water it somehow goes appart (picture). Does anyone has idea why? And what could be a problem?
Hi, what embedding medium do you use?
I am only familiar with cryosection - it sometimes happens with me -
In that case I would say that the tissues are overdried (!) - before sectioning...
Tell us more about the protocol you are using - what kind of fixative have been used ? alcohol? concentration is essential, the same is true for Paraformaldehyde
Hi, are these FFPE sections? If so, are you not using any adherent to fix the sections on the microslides? I used a thin smear of egg albumin and glycerol (1:1 v/v) to fix 3-4 micron thick sections of paraffin embedded Bouin's fixed gonadal tissue. And it worked perfectly.
The protocol is: taking out the brain, cutting it in sagittal line in two halfs, and then puting it in 4% buffered paraformaldehyde, 4 hours at the room temperature, and over night in fridge, after that it goes to sucrose for at least 24hours.
Embedding protocol I don´t have because it´s done by pathologist which I suppose are experts in this field. Paraffin embedded tissuse is returned to me and I put it in paraffin blocks. Then the blocks are going into - 20degrees and then I do slicing.
I am fixing slides with pertex mounting medium.
1.5 micron is really very thin - do you need this? OTOH, it seems you slices come apart into pieces - is the tissue of the quality you need? In that case, your embeddingmedium and handling may be the sources.
OK. If the brain tssue you are slicing is paraformaldehyde fixed and paraffin embedded, the intermittent exposure of the tissue to sucrose solution (cryoprotectant) is essential?. The problem of sliced sections /tissue going apart when taken on microslide (shown in the picture) may be related to need of use of appropriate adherent and also the procedure of embedding especially paraffin infiltration step. Check it with your pathologist. If you are keen of having 1.5 micron sections for histology you may go for semi thin slicing and TB staining with epoxy resin embedding. All the best!
I hope you are cutting the blocks of tissue fixed in Bouin's fluid and embedded in paraffin.
If the tissue is preserved in Bouin's for may days it becomes brittle and hence can come out like that.
Secondly, cutting 1.5 µm on microtome is not recommended because a simple rotary microtome is not suitable for cutting thin sections (1-2 µm).
Thirdly, before you take the sections on the slide, you need to apply a very thin film of Mayer's albumin onto the slides, which acts as an adherent and keeps the sections attached to the slides when warm water is put on the sections to spread them.
I think, most probably the problem that you are facing is due to non-application of albumin onto the slide.
Hope this will help you in sorting out your problem
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