To check the expression of my virus (pAAV-hSyn-DIO-hM3D(Gq)-mCherry (AAV9)—(Catalog # 44361-AAV9)) in the brain, I used IHC according to the following protocol. After confocal imaging, I expected to see cells of uniformly greenish colour. However, in some of the brain sections, the neurons appear red, while in other sections from the same slide, they appear green.

I know that the native fluorescence of the virus expressing mCherry is red, but after following the protocol below, shouldn't all the labeled cells be green due to the use of Alexa Fluor 488 as the secondary antibody? Does anyone know what might be causing this discrepancy?

Protocol:

1. Mice were anesthetized with isoflurane and perfused transcardially with ice-cold 0.1M PBS buffer (pH 7.4), followed by 4% paraformaldehyde (PFA). The brains were then removed, stored in PFA for 5 hours at 4˚C, and transferred to 15% sucrose at 4˚C for 24 hours, followed by 30% sucrose at 4˚C for another 24 hours.

2. The brains were embedded in OCT compound and cut into 50 microns coronal sections.

3. Brain sections were washed with PBS 3 times for 5 min per wash

4. Sections were incubated in 1ml (per well) 1% Triton X-100 in 1:1 ethanol + 0.01M PBS to permeabilize them for 30 min

5. Washed with PBS 3 times for 5 min per wash

6. Sections were incubated in 500µl primary antibody, Rabbit mCherry Polyclonal Antibody (Thermo Fisher, Catalog # PA5-34974; (1:1000), diluted in blocking buffer (3% normal donkey serum in 1% Triton 100-treated PBS) overnight at 4 °C.

7. Washed with PBS 3 times for 5 min per wash

8. Sections were incubated with 500ul secondary antibody 1:1000 Donkey anti-rabbit Alexa Fluor™ 488, Invitrogen™) in blocking buffer at 4 °C for 90 minutes.

9. Washed with PBS 4 times for 5 min per wash

10. Mounted sections onto the slides with Vectashield mounting medium and coversliped.

The images were taken using a Zeiss microscope at 2.5x magnification (I just scanned the slide).

I would be grateful if anyone could help and give me some advice.