Greetings!
Recently, I observed images that looked a bit suspicious to me. I have 2 groups of mice. 1) with the [AAV-hSyn-mCherry] virus and 2) with [AAV-hSyn-hM4Di-mCherry] injected in the cortical region. I just want to know if the DREADD works. So, after CNO administration, I performed IHC staining for DAPI, cFos, and mCherry (mCherry was visualized with antibodies, not by autofluoresence). I noticed strange neurons when observing samples. Some of the images have highly co-localized cells. Cell shapes seem a bit abnormal as well (in my opinion). In turn, the cFos+ cells far from the injection site are roun-shaped with good borders. What is the reason for such high colocalization? Could these cells be considered double-positive? Could they be considered in principle?
First, I thought it was a cross-reaction, but not all of the mCherry+ cells were cFos+, and vice versa, not all of the cFos+ cells are mCherry+. Could this be caused by some (if any) effects of AAV? The virus was injected many months ago, and I suppose mice have completely recovered.
I will be very grateful for any help!
The details about the IHC. The primary antibodies were taken from different species: rabbits and goats. The secondary antibodies were correspondent, anti-Rb, and anti-Gt. All antibodies were incubated with a normal donkey solution (1%). The primary Ab were incubated for 24 h at 4 degrees. Secondary: 1 O/N at 4 degrees. No special staining for cross-reactivity was conducted.
You can find examples of images attached. There is .tiff file that could be used if anyone wants to change some parameters or analyze something. It is a sample from the control group that received AAV-hSyn-mCherry.
Legend:
Cyan: cFos
Magenta: mCherry
Blue: DAPI
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