[Histonet] C3 and C5b-9

[Fischer, Dolores]

Hi fellow histonetters,

I am trying to work out an assay for complement 3 and C5b-9 detection in rodent.   I have moved on to mouse, but have done some work with rats also.  I haven't felt all that successful in my endeavors.   In mice, the tissue of interest is kidney and have thus far proceeded in the following manner:   Section frozen kidneys, place on + slides, air dry overnight, fix in chilled acetone 10 min, proceed with a standard ABC protocol.  I have found rabbit poly from Abcam against C5b-9 in mouse and two anti-C3 antibodies from Thermo against mouse.    My results haven't been all that great and the tissue often comes off the slides, so my results are ugly and frustrating.   Also, I can only go by what I am told and that is that my kidneys should be expressing complement, so I don't really have a good control.

Anyone out there familiar with C3/C5b-9 detection in rodents who can offer any advise or suggestions?  We would like to stay away from fluorescent procedures if possible.  Any thoughts as to why I might be losing my tissue off the slides?

Thank you for your help,

Dolores
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