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Author Topic: Double Staining  (Read 7415 times)

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Offline immunologist

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Double Staining
« on: July 21, 2010, 04:28:53 PM »
Hi,

I stained neurons with TUJ 1 (neuronal marker) and OTX (nuclear trascription factor) using the following antibodies:

Primary AB: TUJ1 (mouse IgG) and OTX2 goat (do not know IgG or IgM)

Secondary AB: Alexa 488 goat anti mouse (IgG) and Alexa 555 donkey antigoat (IgG)

My problem is that signals in both channles look the same. Axons are stained both in red and green. I expect OTX2 only in nucleus. Why is this?

I used FCS for blocking.

Any hel is highly appreciated.

Thanks

immunologist





Double Staining
« on: July 21, 2010, 04:28:53 PM »

Offline CanuckPhD

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Re: Double Staining
« Reply #1 on: July 22, 2010, 06:51:51 AM »
Did you perform an IgG control? You may be seeing background staining. We often have issues with the brain having autofluorescence, especially if the tissue comes from an old subject. We usually use sudan black to reduce this.

Offline funk.106

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Re: Double Staining
« Reply #2 on: July 22, 2010, 09:24:10 AM »
Also, this may be obvious but are you doing anything to reduce crosstalk between the fluorescent signals when you're capturing the image--like sequential scans for instance. I ask only because I had an issue with that once where I thought I had awesome colocalization....and then realized that it was a little suspicious that I ALWAYS had awesome colocalization and figured out that I had set up the sequential scan but then closed the box, so it wasn't actually doing the sequential scan.

Offline immunologist

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Re: Double Staining
« Reply #3 on: July 22, 2010, 03:21:41 PM »

Thanks a lot for the reply.

I did not do a control. Had only one culture dish with cells. It does not look like background flourescence - astrocytes are not stained.

Do not look like an imaing problem.

Is it becuase I used OTX2 GOAT primary AB and  Alexa GOAT anti-mouse secondary? Cross reaction?

Regards,
immunologist






Offline CanuckPhD

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Re: Double Staining
« Reply #4 on: July 23, 2010, 01:57:24 AM »
Without the relevant controls it is very difficult to determine the problem.

Are your secondaries absorbed so they don't cross react with other IgGs? That is does your goat 488 only recognize mouse and not donkey?


Offline Yao

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Re: Double Staining
« Reply #5 on: July 24, 2010, 06:55:46 AM »

Thanks a lot for the reply.

I did not do a control. Had only one culture dish with cells. It does not look like background flourescence - astrocytes are not stained.

Do not look like an imaing problem.

Is it becuase I used OTX2 GOAT primary AB and  Alexa GOAT anti-mouse secondary? Cross reaction?

I think this is very possible.
Regards,
immunologist







Re: Double Staining
« Reply #5 on: July 24, 2010, 06:55:46 AM »