Autofluorescent neurons

bromodomain · Post by **bromodomain** » Fri Jul 08, 2011 5:41 am

I am working on a certain cell surface receptor expressed in ganglial neurons and the method of labelling is based on double immunofluorescence. My secondary antibody is conjugated to Cy3 (indocarbocyanine) meaning that I get red fluorescent signal wherever my receptor is found (plasma membrane). The tissue was frozen in O.C.T. compound and sectioned with a cryostat at 14um thickness.

Normally I wouldn't expect to get any significant signal when I switch to the FITC filter but this was not the case with that sample. I noticed that there was a strong autofluorescent signal situated around the nucleus and I have no idea what it might be.

Carl Zeiss Axioplan
EC Plan-NEOFLUAR 40x/0,75

This is a composite image of a neuron showing the perinuclear autofluorescent signal (yellow-greenish colour around an empty space where the nucleus should be).

I also tried to do some structured illumination imaging with OptiGrid but I think I'm doing something wrong with the calibration since I keep getting these lines from the OptiGrid panel.

Sorry for the quality but I couldn't upload them as TIFs which is their original format.

Has anyone seen such autofluorscent signals in nerve tissue (or other cell types)?

Cactusdave · Post by **Cactusdave** » Thu Jul 14, 2011 11:41 am

Only just back from a trip and seen this. I used immunofluorescence in my research for a number of years when I worked professionally as a cell biologist. True autofluoresence, as opposed to non-specific binding of the second antibody bearing the fluorophore, or spill over of the fluorophore fluorescence spectrum, was something I saw from time to time. The phenomenon was commoner in some cell types than others, frequently located in the perinuclear area, but seldom a problem as it was usually weak compared to the target signal. There is a nice article on the subject of cell autofluorescence here with suggestions on how to deal with it if it becomes an issue. http://www.uhnresearch.ca/facilities/wc ... scence.pdf

bromodomain · Post by **bromodomain** » Fri Jul 15, 2011 2:45 pm

Thanks, Dave!
I find this article particularly useful as a large portion of my samples exhibit autofluorescence which is a major set-back for my experiments.
I tried bleaching my samples but I suspect that the intense lighting rendered the target receptors unrecognisable by the primary antibody.
I'll continue to explore new approaches to solve the problem.

bromodomain · Post by **bromodomain** » Sun Aug 07, 2011 8:59 am

Hey Dave. After a bit of experimenting I decided to proceed with optimizing a Sudan black B staining protocol.

SBB worked at 0.3% (w/v) in 70% EtOH with 4.73% NH3 (v/v) but I got black precipitates when looking at my nerves with the DIC setup. Somewhere I read about the use of 2-(N-morpholino)ethanesulfonic acid and EGTA to reduce the amount of SBB precipitate.

The problem is I can't find 2-(N-morpholino)ethanesulfonic acid in the lab. I did find some MOPS which differs by only a single methylene group (instead of ethanesulfonic its propanesulfonic).

I've been busy with my main project lately but this is something I'd get on with after I'm done with my report and presentation and all that.

Did you ever had to remove autofluorescence?

Cactusdave · Post by **Cactusdave** » Sun Aug 07, 2011 12:09 pm

We are getting into some technical issues here which may be of limited interest to other forum users, so I'll reply by PM next week if that's OK.