*****
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 Doug,


I have a user that has dropped Dapi and swears by Alexa405. Used on an LSM880 it gives great results, even in wholemounts in PBS (no antifade). I didn't believe it until I watched a few scans. Best used for your more abundant antigens but works surprisingly well.


good luck


Dale


http://www.ucl.ac.uk/ich/core-scientific-facilities-centres/confocal-microscopy


________________________________
From: Confocal Microscopy List <[log in to unmask]> on behalf of Cromey, Douglas W - (dcromey) <[log in to unmask]>
Sent: 21 August 2018 00:06:28
To: [log in to unmask]
Subject: recommendation for a "blue" secondary fluorochrome

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
LISTSERV 16.0 - CONFOCALMICROSCOPY List at LISTS.UMN.EDU<http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy>
lists.umn.edu
[log in to unmask]: listserv archives. confocalmicroscopy



Post images on http://www.imgur.com and include the link in your posting.
*****

Is there a better or preferred "blue" fluorochrome? Mostly this is a widefield fluorescence question (using a DAPI cube), but there is the possibility for using a confocal with a 405nm laser line.

I'm working with a group that is trying to work around a frustrating autofluorescence issue in liver tissue. What we ended up doing on an earlier project was not using any secondaries that fluoresced in the green channel (no fitc/AF488/GFP) and instead used the green channel on our widefield microscope as a control, ratioing this image so we could subtract it out of the other channels to remove some of the broad autofluorescent background. This worked nicely. In the published images we showed nuclei (blue) and two specific stains using AF568 and AF647, with reduced background autofluorescence.

The PI now wants to have labelling with three specific fluorochromes, and I am advocating using the green channel as the generic "everything lights up" channel for context, so we can hopefully skip the Hoechst 3342 or DAPI. Since we won't be using DNA dyes, is there a good blue fluorochrome that can be used as a secondary for specific labelling of a marker found on an intracellular organelle (other than the nucleus)?

Thanks,
Doug

------------------------------------------------------------------------------------------
Douglas W. Cromey, M.S. - Associate Scientific Investigator
Dept. of Cellular & Molecular Medicine, University of Arizona
1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

office:  LSN 463              email: [log in to unmask]<mailto:[log in to unmask]>
voice:  520-626-2824       fax:  520-626-2097

http://microscopy.arizona.edu/learn/microscopy-imaging-resources-www
Home of: "Microscopy and Imaging Resources on the WWW"

UA Microscopy Alliance -  http://microscopy.arizona.edu<http://microscopy.arizona.edu/>