[Histonet] Re: Histonet Digest, Vol 119, Issue 20

[Tyrone Genade]

Hello Allyse,

Some questions to your question:

> Date: Tue, 15 Oct 2013 14:43:32 -0400
> From: Allyse Mazzarelli <allyse124 <@t> gmail.com>
> Subject: [Histonet] Autofluorescence on Retina Tissue

> I've consistently run immunofluorescence on pig retina and I seem to have
a
> severe case of autofluorescence/background.

How are you fixing the tissue?

Are you using NH4Cl, 0.1 M glycine etc... to quench auto-fluorescence?

Where do you see the auto-florescence? Which layers of the retina?

Do you have a negative control slides (no primaries or secondaries as well
as no primaries with secondaries)? What do they look like?

What type of microscope are you using? Confocal?

Are you using a nuclear stain such as hoechst?

In my experience there is normally a lot of auto-fluorescence in the retina
to start with. Fixation tends to make this worse... Using 0.1 M glycine
helped me a lot in reducing this problem but the retinal pigment epithelium
layer and the chorion behind it would still auto-fluoresce terribly. The
former is due to lipofuscin so maybe a H2O2 blocking step may take care of
it... Anyone tried? But now I digress... Unless you have negative controls
you don't know what the source of the auto-fluorescence is. Do you have
controls, what do they look like?

I was lucky, as I was working with a confocal and could tune out a lot of
the background fluorescence. If you work with a normal fluorescence
microscope you may have issues, especially if you have tick sections.

IF you are using hoechst as a counter stain for nuclei you may be using too
much of the dye. I had to use as little as 0.5 ug/mL for fixed section
before I stopped getting bleed-through into other channels from the
Hoechst. It showed up in the green and red channels.

Hope this helps...

--
Tyrone Genade
http://tgenade.freeshell.org
email: tgenade <@t> gmail.com
tel: +27-84-632-1925 (c)
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