I got some brains (P1 and E.17 mouse brains) from a collaborator who already fixed them in formaldehyde in 2015, and then transferred to 70% ethanol for storage since then (almost 2 yrs).
I rehydrated them in 50%, 30%, PBS, and then in 30% sucrose/PBS at 4C until they sank to the bottom of the flask. Then, I embedded in OCT and the brains were cut using a cryostat at -24°C at a thickness of 14 um.
After immunofluorescence staining, using GFAP, NeuN, Ki-67 and Sox2 there is no difference between my negative controls and my samples. Seems no positive signal is in the tissue.
I have used antigen retrieval buffers: either citrate or sodium citrate solution, but the result is similar as before, not a positive signal. However, when I used a fresh brain tissue (same age) all antibodies worked.
I was wondering if someone knows a protocol or a kit, I could use in order to make my immunofluorescence staining works. Thank you in advance.
Elizabeth, thank you for your suggestion. I will try to get new tissue.
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