Methods and Techniques Discussion > Immunofluorescence (IF)

Issue with microglia staining on fresh-frozen acetone or ethanol fixed tissue


I would like to establish a quick staining for microglia on fresh-frozen unfixed rat brain tissue. The eventual aim is to selectively isolate microglia for RNA extraction with Laser capture microdissection.

We have very good and consistent results in 4%PFA-perfused and overnight-fixed frozen rat brain tissue with Iba-1 antibody by Wako. However, if possible I would like to use non-PFA-fixed tissue for better RNA yield. I unexpectedly run into troubles by trying to stain for Iba-1 in fresh-frozen unfixed rat brain tissue, postfixed in either acetone or ethanol (see details below).

Does anyone have experience with Iba-1 or an alternative microglia-specific staining (e.g. nucleoside-diphosphatase (NDPase), CX3CR1, CD68,...) in ethanol or acetone-fixed rat brain tissue?

Thank you very much!



A P40 rat was quickly perfused with Saline, the brain immediately removed and frozen in OCT/isopentane. After 2 months storage at -80C, 10um sections were cut at -20 and sections mounted on superfrost plus slides. Slides were kept at -80C until use (1 week). Fixation included -20C or RT ethanol or acetone from 30 sec to 10 minutes. Blocking with 10%BSA was not showing any improvement. Iba-1 antibody (Wako) was applied overnight at 4C 1:800 in 2%BSA, 0.1%Triton (the latter can probably be skipped if fixation media is organic). After washing, secondary fluorescence antibody was incubated 1:500 in 2%BSA, 0.1%Triton for 1hr at RT. Staining resulted in high background signal favoring binding to any kind of nuclei while extremely faint microglia staining could be observed if tissue was fixed for 30sec in ethanol. A concomitant GFAP-staining was working well. A negative control with secondary Ab incubation did not show any unspecific staining.


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