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

[Andrea Marion]

Hi Britt,

There are many many fluorophores you could choose from, including quite a
few that do not overlap with GFP or rhodamine. Here is a partial list:
http://flowcyt.salk.edu/fluo.html

To set up your staining, you will want to first identify a primary
antibody that labels the apoptotic cells you are interested in. Many
people use cleaved-caspase-3 as a marker of apoptosis. I don't know if
this would work in your situation or not. Don't pick a goat primary, since
your ChAT antibody is from goat. Based on what species your primary
antibody was generated from, you will then want to find a secondary
antibody against the primary species that is conjugated to a fluorophore
whose excitation and emission spectra do not overlap with GFP/rhodamine.
This should be easy to find - check Jackson Immunoresearch for the Dylight
series or Invitrogen for Alexafluors.

Your choice of fluorophore is limited mostly by what filters your
microscope has. You need to check that the filter specifications match the
emission spectra of the fluorophore you want to use. I bet you probably
have a DAPI filter; if so, you could probably use a Pacific Blue
conjugate.

Andrea Marion
Graduate Student
University of Illinois at Chicago


----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)
_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet