Hello All,
I have spent the last few months troubleshooting my rat brain IHC. I am new to immuno in mammalian tissue and cobbled together a protocol from the literature and extensive reading of the histonet archives. I'm still not very happy with my results and was hoping that you might be able to weigh in with opinions and/or suggestions.
I am looking at wtGFP (unfortunately I'm stuck with the wild type) and want to combine it with IHC using the red and blue channels. My major problem has been extreme autofluorescence of the sections in the green channel. I have managed to get rid of the overall green background using a sodium borohydride treatment before antibody application and the sparkly kind of autofluorescence (lipofuscin-like?) with CuSO4 after antibodies. I am quite happy with the reduction in autofluorescence, however, the treatments are pretty hard on my free floating sections and many of them end up looking pretty beat-up. Also, no-one else seems to need these treatments and I wondered if I'm doing something wrong somewhere. The only other explanation I can think of is that the wtGFP is less bright and so I can't turn down the background as much when imaging?
Early in my troubleshooting I reduced fixation to perfusion with 400ml of 4%PFA followed by 200ml of 10%sucrose in PBS. The brains go straight into 30%sucrose with no post-fix. I'm having a lot of trouble now, though, with slicing and section integrity so I think I may need to go back to a post-fix, especially since the borohydride and copper seem to work for getting the background down. A lot of my sections are very fragile, sticky and sort of melt-y making me think that I've got insufficient fix.
I know I'm going on here so I'll just summarize my protocol and ask you to please comment or suggest possible problems or solutions.
Perfuse rat via cardiac puncture; flush with PBS (I was doing 200ml but will reduce this next time to 20-50ml); fix with 400ml PFA; flush with 10%sucrose in PBS; remove brain and place in 30%sucrose refrigerated until it sinks. Freeze brain in 30%sucrose at -80. Cryosection 30um sections at -17degrees into cryoprotectant (Glycerol/Ethylene glycol/PBS); store at -20. For IHC wash in PBS; treat sodium borohydride, wash PBS, preblock with goat serum; primary overnight; wash and then secondary overnight; wash; CuSO4 treatment; wash; mount on superfrost in water and coverslip with Prolong Gold.
Thanks very much I appreciate your input.
Nadja
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Nadja Spitzer, Ph.D.
Cell Differentiation and Development Center
Department of Biological Sciences
College of Science
Marshall University
1 John Marshall Drive
Huntington, WV, 25755
email: spitzern@marshall.edu
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