Hi Everyone,
In the past couple of months, I have been trying to do in-situ hybridisation of some mounted fish brain slides, and I have had huge problems on day two after the 50% formamide:2xSSC washes.
We routinely prepare the solution for day two on the previous day. When we open the 2xSSC: 50% Formamide solution jar (in the oven at 60C), we notice a distinctive smell of Ammonia, sometimes quite strong other times not so much. Every time this phenomenon is present, the slices will eventually peel off from the slides, and we lose all the data.
Has this happened to anyone? Does anyone know what might be the reason behind it?
We use 50%Formamide/2xSSC as a prehybridization solution at 37°C.
And I prepare it freshly on the same day when I want to use it. Normally I prepared the solution at first before I start with my ISH because it has to warme up to 37°C before I start with the prehybridization step. Taken together: reasons why you lost your sections could be: 1. Temperature of the Formamide/2xSSC solution, the freshness of the used solution the kind of slides you have used for mounting your sections. I recommand to Superfrost plus slides.
Hi Ute,
Thank you so much for your reply. We use super frost plus slide; the only difference is that we incubate our probes at 60C and do the first washes of 50% Formamide: 2x SSC at 60C before doing some 2xSSC washes at 37C.
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