[Histonet] re: triple immunofluorescence for apoptosis (bluefluorophore?)

[Anatoli Gleiberman]

Bill,
There are plenty of secondary conjugated with blue fluorophore (check JacksonImmunoresearch or Invitrogen catalogs), but usually these fluorophores are not very good, signal is dull and long exposure burns signals not only in blue channel, but in red and green as well. If your microscope is equipped with filters for deep red channel you can use Cy5 or DyLight647 from Jackson or AlexaFluor647 from Invitrogen. You will not be able to see the signal by eye, but your digital camera should be able to register it - and leave blue channel for DNA dyes such as DAPI or Hoechst.In this case, you will have four color fluorescence. 

Anatoli Gleiberman, PhD
Director of Histopathology
Cleveland Biolabs, Inc
73 High Street
Buffalo, NY 14203
phone:716-849-6810 ext.354
fax:716-849-6817
e-mail: AGleiberman <@t> cbiolabs.com

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Britt Tracy
Sent: Thursday, March 17, 2011 12:30 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] re: triple immunofluorescence for apoptosis (blue fluorophore?)

 Good morning!

I'm looking for advice on an antibody conjugated with a blue fluorophore
that could be used to tag for apoptosis (on p75 receptors on cholinergic
neurons in the rat basal forebrain).

We use a Goat X ChAT primary (1:100) and a Rhodamine-conjugated Donkey X
Goat secondary (1:250) on the slices.

The slices already contain GFP expressed by the viral vector we infused into
the basal forebrain.

So, when we image the sections we already have a green fluorophore and a red
fluorophore. Is there a blue fluorophore tag for apoptosis that anyone has
used which would be biochemically compatible with the antibodies above?


Is there another fluorescent option? Internet-searching and
literature-reading has not gotten me anywhere.

Our scope is a Leica DM4000B equipped with a Leica DFC 425C camera.



Thanks for your time; any advice or insight would be much appreciated.


Sincerely,

Britt Tracy (undergraduate researcher at Temple University)
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