Hi there,
I am trying to measure dendritic spines from mouse fixed tissue, specifically, I am looking at the prefrontal cortex. I injected the PrL with a lentivirus to knock-down my protein of interest and now I would like to measure any differences in spines. I can't use Golgi staining because it will get rid of my transduction. I tried IHC but all I see is fluorescent cell bodies and not dendrites. Has anyone tried IHC to measure dendritic spines in the PrL? If so, is there a way to improve the IHC so the dendrites are visible?
Mouse was perfused with PBS and 4% PFA
Brains cryoprotected with 30% sucrose
Frozen Sections at 20 um.
My protocol for IHC is
10% NHS blocking soln
Anti-GFP(1:3000) in 1% NHS blocking soln overnight at 4 degrees
Wash 3x15 mins
Alexa-Fluor 488
Imaged at 60X on the confocal.
I understand IHC would not be afidable ever. You must test other staining like Cox-Golgi.
Dear Nadine,
Any chance you could change your GFP reporter to membrane-targeted version. This would likely homogenize the GFP signal trough the entire neuron including dendrites and spines. You could also bleach your samples in peroxide solutions to reduce background before GFP labeling. Tissue clearing protocols may also be a strategy to consider.
Cheers,