I would like to perform immunostaining and in situ mRNA hybridization on whole-mount large insects (e.g., not thins sections, but can dissect parts). I am working with larval, pupal and adult stages, not embryos. I know the insect cuticle is hard to permeabilize, and tends to show autofluorescence and to bind non-specifically with many probes. I would appreciate any protocols or tips on fixation, preparation, and staining, that could help me work out a suitable procedure to obtain informative immunostains. Thanks!
Use picric acid 10% as fixative for whole insects, and for its soft parts you can use 10% formaldehyde for 10-12 hrs fixation. Wash it running water. You can use two contrasting stains for staining purpose, Hematoxylin and eosin are good combination for differentiation of tissues and cells. Use down and up series washing methods to find clarity in each subject.
Hi Eduardo,
Probably, my answer arrives too late.
I have experience with whole mount ovary and embryo of cockroaches and one tip I can give you is in order to avoid autofluorescence of tissue (ovary has a lot) or at least that it does not disturb your labeling. We are using now far red fluorofores (as Alexa fluor 647 or Cy5) that are not visible under microscope (eye view) but if you have the proper laser it is a good option.
As a rutine we fix the tissue with PF4%, but you can try also a combination with carnoy (it works well to nuclear staining?.
I know a great product to become clear the tissues named ScAle (8M or 4M, check it), but was too aggressive for our samples, you can try i.t.
Regards
Hi Eduard,
I mentioned below my protocols with whole mount method for observing the early embryonic markers of Galleria mellonella and hope its useful for you.
http://docsdrive.com/pdfs/academicjournals/je/0000/88517-88517.pdf
Best wishes
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