zebrafish autofluorescence

[张莉莉]

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Dear all,

Since our confocal microscope was broken, now I have to use inverted fluorescent microscope. Now the problem I have is that after I fixed zebrafish embryos with 4% PFP overnight in the fridge and did the immuno labelling, the embros had a lot of autofluorescence. Could somebody know how to advoid and get rid of it? Thanks a lot!

Best regards,

Lili

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[张莉莉]

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sorry it is 4%PFA. a mistake

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在2016年09月23日 14:55，[hidden email] 写道:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Dear all,

Since our confocal microscope was broken, now I have to use inverted fluorescent microscope. Now the problem I have is that after I fixed zebrafish embryos with 4% PFP overnight in the fridge and did the immuno labelling, the embros had a lot of autofluorescence. Could somebody know how to advoid and get rid of it? Thanks a lot!

Best regards,

Lili

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[Zdenek Svindrych-2]

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Lili,

are you sure it's autofluorescence? Have you compared you immunolabelled embryo with a similar one (fixed the same way) without the fluorescent antibodies?

Of course, if your sample is thick, the widefield collects much more autofluorescence from the sample than a confocal, but it also collects all the out-of-focus fluorescence! In some densely labelled thick samples you can see great details in a confocal, but just a bright homogeneous "sea of fluorescence" in a widefield.

On the other hand, depending on the stage of the embryos, there may be a lot of autofluorescence from the yolk. You can try to cut away the most autofluorescent parts, you can try to "clear" the sample - permeabilize and remove lipids, some people try to bleach the autofluorescence (after fixation, with some strong light, or even Californian summer daylight was reported to work :-), if you suspect the PFA fixation is the source of your autofluorescence, you may try to use sodium borohydride (reduces the remaining aldehyde groups).

Different approach would be spectral unmixing (if it's really autofluorescence that bothers you, you may excite and detect it at different wavelengths than your fluorescent antibody) or deconvolution.

Or you may switch to other color of your fluorophore where the autofluorescence is weak....

Good luck.

zdenek

---------- Původní zpráva ----------
Od: 张莉莉 <[hidden email]>
Komu: [hidden email]
Datum: 23. 9. 2016 8:56:34
Předmět: zebrafish autofluorescence

Dear all,

Since our confocal microscope was broken, now I have to use inverted fluorescent microscope. Now the problem I have is that after I fixed zebrafish embryos with 4% PFP overnight in the fridge and did the immuno labelling, the embros had a lot of autofluorescence. Could somebody know how to advoid and get rid of it? Thanks a lot!

Best regards,

Lili

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[张莉莉]

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Dear Zdenek,

Today I tried to fix embryos with 4%PFA for 2 hours at room temperature instead of over night at the fridge. And there are less autofluorecence. 

Best regards,

Lili

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在2016年09月23日 15:52，[hidden email] 写道:

***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****

Hi Lili,

are you sure it's autofluorescence? Have you compared you immunolabelled embryo with a similar one (fixed the same way) without the fluorescent antibodies?

Of course, if your sample is thick, the widefield collects much more autofluorescence from the sample than a confocal, but it also collects all the out-of-focus fluorescence! In some densely labelled thick samples you can see great details in a confocal, but just a bright homogeneous "sea of fluorescence" in a widefield.

On the other hand, depending on the stage of the embryos, there may be a lot of autofluorescence from the yolk. You can try to cut away the most autofluorescent parts, you can try to "clear" the sample - permeabilize and remove lipids, some people try to bleach the autofluorescence (after fixation, with some strong light, or even Californian summer daylight was reported to work :-), if you suspect the PFA fixation is the source of your autofluorescence, you may try to use sodium borohydride (reduces the remaining aldehyde groups).

Different approach would be spectral unmixing (if it's really autofluorescence that bothers you, you may excite and detect it at different wavelengths than your fluorescent antibody) or deconvolution.

Or you may switch to other color of your fluorophore where the autofluorescence is weak....

Good luck.

zdenek

---------- Původní zpráva ----------
Od: 张莉莉 <[hidden email]>
Komu: [hidden email]
Datum: 23. 9. 2016 8:56:34
Předmět: zebrafish autofluorescence

Dear all,

Since our confocal microscope was broken, now I have to use inverted fluorescent microscope. Now the problem I have is that after I fixed zebrafish embryos with 4% PFP overnight in the fridge and did the immuno labelling, the embros had a lot of autofluorescence. Could somebody know how to advoid and get rid of it? Thanks a lot!

Best regards,

Lili

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