1) If I have an intracellular molecular probe (647 nm) perfuse the trasgenic animal and paraffin embed the tissue will I be able to see the fluorescence with a confocal of fluorescence microscope?
2) If I had the same sample as described above, you I be able to use primary antibody (e.g., anti-Iba1) and an Alexa fluor secondary on paraffin-embedded tissue?
If the probe can be fixed to the tissue so long as it's kept in the dark it should be fine. If it's not something that's fixable it will probably wash away before you get to image it.
With regards to antibodies check the manufacturers spec sheet and it should tell you if the primaries are compatible with paraffin-embedded tissues. Alexa secondaries are.
Hi Norelle, Howard gave you a sound advice. Re your point 2 - there are several primary antibodies on the market if you wish to look for Iba1 which work on FFPE material (e.g. abcam & GeneTex) which give excellent results for IHC for light microscopy, so using Alexa tag on a secondary should not be a problem. If you wish to have a double-staining - have your first probe well detected first and consecutive sections stained for Iba1 before you try to combine - the Iba1 needs antigen retrieval, so you might need to modify the second detection slightly (low temp, gentler AR, and longer primary Ab exposure on the bench or in incubator). Good luck with your project.
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