I have a whole chromosome paint for rat X chromosome but so far the staining was unsuccessful using the protocol the supplier provided for inter- and metaphase slides. Hence I am wondering how the protocol needs to me adjusted to suit the use of brain slices? How do the brains have to be prepared before hybridization? Is it even possible to carry out FISH in intact brain slices?
Please tell the community your detailed protocol. What kind of processing (frozen, fixed, paraffin embedded,...). How thick are the slices?
Sure. Usually I perfume with prefix and then PFA 8roughly 20ml). After removing the brain it is left in PFA for a night and in 20% sucrose until it sinks. Then it is rapid frozen embedded in tissue etc and sliced at 20um. Slices are directly mounted on superfros plus slides. Then I apply the ready to use chromosome paint probe, cover and seal, denature slices and probe together at 75°C and hybridize over night at 37°C. Does that help? Many thanks!
As far as you work with formaldehyde fixed tissue you should follow a protocol that has a permeabilization step. For FFPET an incubation with hot Citratbuffer pH6 and digestion with Pepsin is recommended. What specimen-type is the provider's protocol for? Isn't there any permeabilization demanded?
You also have to take into consideration, that for fixed tissue the length of the probe can be problematic. For FFPET probelengths about 200 bp are recommended to be able to penetrate the tissue.
I hope a specialist in this field gives further information.
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