I am attempting to stain the hippocampus of the mouse brain with fluorescent NeuN and Nissl staining (cresyl violet). However, both NeuN and cresyl violet results show empty cells, leading to low imaging quality and making analysis challenging. In this case, could the problem be in the perfusion or the IHC protocol? I'm not sure about the expecting potential causes. Any help would be appreciated. Thank you.
Do you have a DAPI stain? I'm not sure about the phenotype of your mouse model, but it looks like some cells only express the marker in the cytoplasm, the "empty" part of your neurons should therefore be filled with DAPI.
Some more information would be helpful, e.g. how thick are your sections? Did you do cryo or FFPE fixation? What are the details of your staining protocol? What type of image analysis are you aiming for?
I don't think it's the perfusion, since the tissue sections look really good! You could try to add TritonX (0.1 - 1%) to blocking buffer and primary antibody mix to permeabilize the tissue and make sure the antibody reaches the nucleus.
By the way, this is not low image quality. Your Nissl stain is textbook pretty ;-)
Suckert Theresa Thank you for your answer. Below is the staining protocol I have been using. I would greatly appreciate your assistance once again.
[1] IHC
cryosection, 30um, free floating
wash : 10m * 3 times pbs (1ml)
blocking : 1h room temperature (300ul) (0.3% pbs-t and 5% bovine serum)
primary ab : 4'c over night (200ul, MAB377, i did several titer test)
wash : 10m * 2 times pbs (1ml)
secondary : 1h room temperature (400ul) (0.3% pbs-t)
wash : 10m * 2 times pbs (1ml)
mounting with dapi
[2] Nissl staining
cryosection, 30um, slide mounting
100% ethanol 2min
95% ethanol 1min
70% ethanol 1min
water for several times dip (5~7 times)
0.1% Cresyl violet solution for 3min
water for several times dip (3~5 times)
70% ethanol 1min
95% ethanol 1min
100% ethanol 2min
Xylene 1min and then mounting
Additionally, I am attaching the DAPI results here. It can be observed that cells with empty cytoplasm also exhibit only membrane presence in DAPI staining.
Well, that's unfortunate, there goes my theory...
I have never seen that effect in my brain samples (FFPE fixed), so I'm not quite sure what to suggest. Do you do cryopreservation with sucrose? Does pbs-t mean pbs + triton?
Suckert Theresa After fixing the brain with PFA, it is placed in a 4'C refrigerator until it sinks in a 30% sucrose solution. Following sinking, the brain is embedded in OCT compound and stored in a deep freezer at -80 degrees until cryosection.
PBS-T means a solution containing PBS and Triton X-100.
Thank you for your assistance. I plan to study a bit more and try again.
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