[Histonet] Re: Colon whole mounts and counting aberrant crypt foci (ACF)

[Andrew Gray]

Hi John & Histonetters,

I've done a lot of fluorescence staining on whole-mount rat ileum.   The reason the tissue is pinned to the cork (or a balsawood sheet in my case) is so that it is under tension while being fixed.   This produces easily-mountable flat tissue sheets.   Without fixation under tension, the tissue can be quite warped, as you have seen.   

I expect the physical bulk of whole-mount tissues would pose a problem in keeping them adhered to slides in processing.   You may be able to reduce the thickness by dissecting away unwanted layers.   I would suggest you consider free-floating processing, as it is not too difficult.   I use cell culture plates (96 well), with the wells containing the staining reagents.   The whole-mounts are processed through the wells in sequence using a soft paint brush or fine dissecting forceps.

When mounting on slides the whole-mounts are positioned while in your buffer.   Then, the buffer is blotted away and an appropriate mountant is added and the slide coverslipped.   This may be different for ACF staining.

Best wishes,

Andrew Gray
PhD student
Victorian College of Pharmacy, Monash University
Australia