http://confocal-microscopy-list.275.s1.nabble.com/Tissue-imaging-auto-fluorescence-and-bright-dot-like-signal-tp7588326p7588357.html
How long are the perfused brains sitting in fixative or buffer before sectioning? Formaldehyde fixation may reverse with prolonged storage and bits of tissue begin to come apart and absorb label. Worse, bacterial contamination occurs which seems to either ingest labeling components or the fluorophores stick to the bacterial glycocalyx. This may be tested by observing mounted sections under a non-hardening mounting medium and running a short time-lapse to see whether your dots are moving.
> On Jun 7, 2018, at 10:22 PM, Yan, Lu <
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> Hi Tala,
>
> Thanks much for the info. The tissue is from perfused mice. Regarding the dots in non-stained controls, we found clearing using pk and sds reduced the af a lot, e.g. the raw intensity dropped from 6~7k to ~1k whereas the probe signal can reach to 6k or so. But these dots are different under different channels (spatial distribution also differed). blue e.g. alexa 488, and green e.g. cy3 have the strongest signal, and they seem to be different dots under different channels.
>
> Thanks,
> Lu
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:
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> Sent: Thursday, June 07, 2018 4:02 PM
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> Subject: Re: Tissue imaging auto fluorescence and bright dot-like signal
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> Hi Lu
>
> It might be the methanol fixation. That could contribute to AF quite a bit. Also is the tissue from perfused animals? If not, could it be red blood cells/blood clots? They AF like crazy. Lipofuscin is a huge issue in imaging human brain but haven't come across as a problem in rodents (although perhaps aged rodents might be a different story). Lastly, although this may not be relevant here as you said the same dots were present in non-stained controls, too much fluorophore on the sample can often lead to bright dots like that.
>
> Hope this may provide some leads for you to investigate.
>
> Best wishes
> Tala
>
>
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> Friday, June 1, 2018, 1:38 PM +1000 from
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> Dear listers,
>
> I have been imaging mouse brain tissue samples for the last a few days using multiple wavelengths laser as illumination (from 405 to 750 nm). Initially, the targeted genes in the brain slice were labeled with different dyes, e.g. Cy3, Cy5, and Cy7 etc., and I observed quite bright dots in corresponding channels, and thought they were RNA molecules. However, further examination showed that those dots showed up even in 'plain tissue' samples (no probes at all), and they virtually showed up in almost all fluorescence channels.
>
> The intensity was quite high that we could not distinguish the fluorescent probes signals. The plain tissue was snap-frozen and sectioned, and fixed with just methanol on coverglasses. I had tried different part of brain, different mouse, and they all showed similar results, i.e. bright dots cross the tissue sample, sometimes dense, sometimes sparse. They also distributed along the z axis, over about 10 um which is the sample thickness. In some region, I can see larger clusters giving much stronger signal. I also tried mouse liver samples, and the signal level was very similar to those from brain samples.
>
> I was wondering if anyone had similar experience in imaging tissue samples. What could be causing this kind of signal? Any input is welcome and appreciated.
>
> Thanks in advance,
> Lu
>