Hi all,
We're having an issue during IHC with celloidin-treated, paraffin-processed human brainstem tissue sections, one that does not occur with our typical paraffin-processed sections. The tissue has a tendency to fall off the slide at the first xylene step (deparaffinization).
We've tried upping our typical Superfrost Plus slides to Gold slides, as well as baking the mounted tissue sections for up to two weeks beforehand in a 60*C oven, to no avail. The primary difference between these cases and others is that the tissue was celloidin-treated before being paraffinized (I can provide more details if needed; writing this on behalf of a colleague). I have read that air bubbles/water between the tissue section and slide during mounting can lead to tissue falling off even charged slides, but I do not believe this was the issue as it seems specific to (and ubiquitous within) these cases.
Does anyone have experience staining celloidin-treated, paraffin-processed human tissue, or some idea of what might be causing this tissue to fall off positively charged slides?
Thanks so much in advance!
In IHC there are many steps of washing with PBS solution and may be lead to fall of sections especially the relatively heavy sections ( treated with colloidin-treated ) .... I was prepared charged slide by my self and they were perfect>>>> try this methods and be careful with steps of rinsing with PBS
Agree with Richard,
Even though the slides are charged, I used to get this problem especially in HRP-chromogen IHC in my case. I used to coat the slides (a very thin layer, I used to use the cover slip the coat the slide as if you are doing a blood smear preparation) with egg albumin. Even raw eggs (need not be a Barnded product) was good enough.
This might sound "off" but are you sure that you are using a charged slide? One of the labs I was in had just that issue - got a batch that wasn't and went ahead as if it they were.
In IHC there are many steps of washing with PBS solution and may be lead to fall of sections especially the relatively heavy sections ( treated with colloidin-treated ) .... I was prepared charged slide by my self and they were perfect>>>> try this methods and be careful with steps of rinsing with PBS
There are two approaches that can be tried. The first as stated above uses an egg albumin fixative to thinly coat the slide. (a clean finger works pretty well).The second is somewhat similar and employs 1 g of gelatine in 1 liter of hot distilled water. When cool add 0.1 g of chromium potassium sulfate. This method can be found in an old book by Boyd G.A. Autoradiography in Biology and Medicine, 1955, Academic Press. You can also try coating your slides yourself with polylysine (www.genome.wisc.edu/pub/reprints/GEC_Slide_Coating.pdf) and not trust the commercially available slides.
Yes, you can try to coat the slides with chromium postassium sulphate + gelatin. This is the best I observed for paraffin sections.
I agree with the collegues above - don't trust to the producers and laboratory technicians, do everything yourself. Tr
y to use the most "fresh" glasses. And heating at the 36-40 C helps too
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