I de-cellularized brain sections and wanted to verify that there were no more cells. I used DAPI to do this since it is specific for nuclei. In my controls (picture A) there are obvious labeled nuclei. In the de-cellularized tissue (picture B), there are no nuclei but it looks like the microvasculature has been coated in fluorescent blue dye. I've attached some pictures. Does anyone have any thoughts on this weird DAPI staining? Thank you!
Which is the exposure of the two pictures? Maybe autofluorescence? Use the same exposure for a and b.
Which DAPI staining protocol do you follow?
Yeah, I agree about exposure time - they look different to me...and both look a little over-exposed. Jan is also right - if the DNA just gets spread out all over then the DAPI will make apparently streaky blue marks. But those are usually far weaker in signal than the "real" thing, so proper exposure controls will remove it. I'm also not surprised you see DAPI in the vasculature, since you didn't disturb those cells. It's hard to tell from your pictures, but it looks like SMC nuclei are stained (as you would expect).
I agree with the above comments and wanted to emphasize the autofluorescence part. The extracellular matrix which is left after decellularization is highly autofluorescent. This is a problem I have as well. I did not finda a good way to minimize it yet and do what the others suggested and turned down the gain.
I fully agree with all the above comment. Exposer time is very important for the flourescent dye. Over- expose can easily kill them. I also agree with Jan, you may remove DNA during your de-cellularization. Good luck with experiment
How did you decellularize the brain? The best troubleshooting option is to run a control - decellularize the way you did, don't stain it with dapi and observe. If it is autofluorescence, it would show up. We will think about workarounds after we know that it is not autofluorescence.
I do gree with this last comment. From your low mag in B it seems that you have a lot of staining (see the band top right). The b2 magnified picture is out of focus but it seems that there are cells insided vessels.
Is staining done after fixation or not? This also makes a difference.
Try to wash carefully.
At what concentration are you using DAPI? It might be either this, exposure or washing. Try a no-stained control. I have gone as up as 1:2000 for dapi
Hi Kimberly,
first of all I would check the concentration of your DAPI-Stock solution!
In my hands a final concentration of not more than 5 µg/ml (DAPI) was enough to get a proper staining of nuclei in brain sections of 25 µm.
If you have used a confocal microscope with a 405 nm laser you can also reduce the band pass filter for emission to minimize unspecific backgroud signals. Having a look at the emission properties of DAPI (Ex. 405 nm) will show you an emission range from approx. 410 nm up 500 nm. Reduce the band pass to a gate of +/- 15 nm around 440 nm could help to reduce unspecific signal wherever they might come from. After this step I would adjust exposure time, PMT and laser intensity to reduce risk of unspecific signals. Start with the positiv control--> proper microscope settings--> checking de-cellularized sections.
It might be possible that the origin of the unsharp pictures with fals positiv signals come from an unproper mounting. To much mounting medium or a sloped cover slip could have the same effect!
Best
Nick
Hi, is there a final answer to this? Thanks!
I will say that is precipitated DNA due to the use of Sodium Deoxycholate.. I have the same structures.
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