Hi Everybody,
I have been setting-up colorimetric in situ hybridization in my lab. For all the genes I am interested in, I have generated DIG-labeled Antisense and Sense Probes, and I have ran some preliminary trials to optimize the technique and make sure that everything is working the way it should. So far, it seems that everything is working nicely: nice signal with all my antisense probes (in the brain areas I was expecting the signal to be), no signal with the sense probes or when no probe was added to the hybridization buffer.
I am now ready to start my experiments. However, before starting I would like to get some feedbacks. I am planning to include to my experiments some negative control for every in situ I am about to run, to make sure that everything went smoothly during each in situ. Given the fact that for each in situ I will have many slides to stain, I was planning to add only one slide per in situ (and thus per animal) with the sense probe and one without any probe, just to make sure of the specificity of my staining for all the slides stained during every in situ experiment.
Is that enough, or should I plan to use for the S probes all the slides corresponding to the adjacent sections I am using for AS probes labeling? I took a look at the literature, and that doesn’t seem so clear… What is the common practice ?
Thanks in advance for your answers and tips !
Marie
One without any probe and one with antisense probe per in situ should be okay.
I agree that one sense and one no-probe control will be sufficient. A positive control does also help if you have access to one, but not everyone can use a positive control if they are not working from an established protocol.
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