Hi there,
Yet another IF-related question. I'm going to be dual-labelling free-floating tissue sections with fluorescent secondary abs. After the final wash steps, I will be mounting sections onto slides using a DAPI medium.
Maybe a silly question but will I have to mount my sections in the dark, or with very minimal light at least so the fluorescence isn't affected?
Or does anyone know if I could mount sections in a dark room with a dark room lamp (does that affect fluorescence?), usually where western blots are developed?
Thanks in advance
Hello David,
No need to mount tissue sections in dark, you can do it in dim light or by switching off all lights in normal room , where some natural light can help you to mount ur sections well. No need to go for dark room or red light. A little light will not quench the fluorescence.
Cheers !
I agree with Ajay Kumar? you do not need mount in in the dark? but the time of loosing fluorescence demnd on the fluorophore yoou are using with your primary antibody!
Like if you use FITC- or TRITC- labelled abs, it will fade faster that ALEXA, mining that morden fluorophores are usealy more stable and less sensitive to the wite light that previous ones/ good luck< natalia
Don't worry too much about mounting in a normally lit (or slightly dim) room.....most secondary antibodies hold up fine in these conditions.
I would, however, add that if you have several wells to mount (e.g. 30-60 minutes per brain * 6-12 brains)... keep the other brains, that you aren't mounting yet, in the fridge (which is presumably also dark, or tent them with foil). This is a better practice than having them all on your bench (in the light at room temp) for the full 3-8h that it can take to mount an entire IF experiment.
Excellent. Thank you everyone! That is such a relief as I have literally hundreds of tissue sections to image!
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