[Histonet] confocal condition for Cy2 and Cy3 IHC on plants

[Mansfield, James]

> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu 
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf 
> Of Moran Oliva
> Sent: Thursday, July 20, 2006 2:09 PM
> To: histonet <@t> lists.utsouthwestern.edu
> Cc: Moran Oliva
> Subject: [Histonet] confocal condition for Cy2 and Cy3 IHC on plants
> 
> Hello all!
> I've just joined this forum. It is very useful. 
> I've started a preliminary assay of Immunohistochemistry on 
> Arabidopsis tissue (Steedman's protocol) using my GFP-protein 
> transgenic plants as a positive control (to confirm the 
> method is working for me).
> The anti-GFP antibodies I used were either from mouse or from 
> rabbit. So when I used the Cy2 goat anti-mouse I needed to 
> use the same emission spectra as for the GFP protein itself-  
> Does anyone knows whether the GFP survive all this process? 
> does anyone has any good advise of detecting the IHC signal 
> and not theprotein GFP signal? the second channel I used was 
> GFP autoflouresence...
> Does anyone know what are the confocal setting details for 
> Cy3 in plant tissue? I've tried to detect the GFP in IHC 
> using rabbit GFP protein, so my second antibody is Cy3 
> conjugated ( I know the excitation and emission spectra, but 
> the autoflourescence is very close to its wave lengths-do I 
> need to use a second channel for autoflourescence in this case too?

Hi Moran,

A good place to find wavelengths for fluorophores is the Curv-omatic
site from Omega Optical:

https://www.omegafilters.com/front/curvomatic/spectra.php

It will show you absorption and emission spectra for almost any
fluorophores you might want to use.

However, having said that, separating fluorescence from your fluorophore
from autofluorescence can be very difficult for most systems.
Autofluorescence emits at almost all wavelengths, and will conflict with
just about any filter set you want to try.

A multispectral imaging approach can help a lot in eliminating
autofluorescence from your samples, with signal-to-noise increases of
100-fold. 

Here is a link to a paper we are publishing in Cytometry later this year
(two issues from now, I believe) that discusses using multispectral
imaging to separate fluorophores from autofluorescence. The examples are
done using our own spectral imaging system (Nuance), but the approach
applies in general.

http://www.cri-inc.com/files/MSI_App_02.pdf

Cheers,

-Jim