Hi,
I would like to use Villanueva staining on bone samples that have already been embedded in plastic (MMA) and sectioned to 10um. Does anyone have a protocol for these 10um sections?
I can find many articles for staining the samples before embedding, but no protocol for handling a thin sample like mine. The manufacturer's protocols assume a bone sample of 2-3mm thick.
Alternatively does anyone know of a more gentle differentiating solution for Villanueva stain? The methanol/acetic acid solution suggested by Sigma-Aldrich is too strong for these sections and they get destained too fast to be evenly destained.
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/1/shh0027pis.pdf
Thanks in advance!
Catherine
Hi Catherine:
For toluidine blue our lab elaborated the following protocol for 5-15 micrometer-thick sections cut from plastic embedded blocks:
1) Put slides for 30 seconds in jar of 40% acetonitrile
2) Rinse slides in jar of 100% ethanol (dip a few times and remove)
3) Rinse in jar of distilled water (dip a few times and remove)
4) Cover bone part of sections with drops of TB stain
5) Leave flat for 15 minutes in humidity box (I use a slide box with some wet paper towels under where the slides are sitting, on top of the slots).
6) Rinse in tap water until water is clear (fill jar with water, dip in, pour out water and refill, dip in, etc.)
7) Lay out paper towels on flat surface. Gently put slides FACE DOWN on the paper towels and cover with more paper towels. Let dry at least 2 hours, under hood.
8) Dip in xylene and coverslip with Permount.
Hi Timothy G. Bromage ,
Thanks for the protocol. I have a couple more questions to help me understand how this protocol is modified for the plastic sections. I assume the sections are still in plastic for this method (i.e. no deplasticization before the acetonitrile, or is the acetonitrile itself deplasticizing the section, at least on the very top surface)? How different is this protocol from your standard toluidine blue e.g. for paraffin sections? e.g. longer stain time, more concentrated stain solution?
I really appreciate your help!
Thanks,
Cath
Hi Catherine:
We only process hard tissues with the mineral intact. We have never demineralized and paraffin-embedded bone before. You got it. The acetonitrile very gently solublizes the surface plastic and allows the stain to penetrate ca 5-10 micrometers. This is quite wonderful on mounted 100-micrometer-thick sections because if you place such a slide over an opaque white plastic sheet, which allows the light to pass through, what you image is only the thickness that is stained (i.e., 5-10 microns). Such sections are so much easier to make and afford many more microscopy techniques than ultra-thin sections.
Thanks! I have seen other protocols with hydrogen peroxide as the first step - do you know if this has the same effect as the acetonitrile, or is hydrogen peroxide more of a etching treatment for the whole sample surface?
I will certainly try your protocol - thanks very much!
You are correct about the hydrogen peroxide. It would have an effect on the matrix, not the plastic.
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