I am doing WMISH experiments and usually get the stained embryos. I am not able to take the picture of my results. When I observe my embryos under microscope it looks like in the picture attached. Should I use any special lightings or is it something with the microscope? I have a NIKON Microphot FXA with attached camera.
Are you using a top or a bottom light source? This can make an enormous difference. For good quality in situ pics use top lighting, and place your embryos on a white background. (Also, take care to adjust white and black levels in your imaging software.)
With early stage embryos aspecific staining of the yolk can also be a problem (especially if you are using NBT/BCIP). If this is the case I'd suggest using BM Purple instead as chromogen.
Hi, I think you only need a translucent base in your microscope and a good support for the specimen (e.g. 24 well plate or ceramic ISH plate). Also it seems that your samples are over stained. No special equipment is needed!
Are you using a top or a bottom light source? This can make an enormous difference. For good quality in situ pics use top lighting, and place your embryos on a white background. (Also, take care to adjust white and black levels in your imaging software.)
With early stage embryos aspecific staining of the yolk can also be a problem (especially if you are using NBT/BCIP). If this is the case I'd suggest using BM Purple instead as chromogen.
I agree with Mate Varga; you need the light source on top of your sample, and a white background under your sample (eg: a white paper sheet). To make the yolk more transparent, you can use mixes of benzyl alcohol/benzyl benzoate 1:2. Be careful with BABB when using alcohol-soluble chromogens (eg: fast red or fast blue).
It actually depends on what you are looking to accomplish. It appears that your problem is a staining and/or lighting problem. Are you lighting from the bottom or from the top? Usually whole embryo mounts such as this need to be less stained and lit from the bottom or sides to give the appropriate effects for documentation. Alternatively you may try a different stain that would be a little less and show the type of structures you are wishing to observe.You may try fluorescent dyesas this would get rid of the extra staining issues as well.
@ Jan Brocher:
Do you stabilize the stacks of the smaller coverslips with anything (like nailpolish) or is this construction stable by itself?
Another problem might be the oben sides of the "bridge". Is it necessary to seal them in order to prevent leaking of the glycerol?
Thanks, best regards
Jörg
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