[Histonet] Primary works with ABC but not IF

[John Steel]

Hi All,

I do agree with titration - try this first.  You may consider pre-blocking before use of the primary antibody.  In my experience, tryamide amplification is expensive, and may not be necessary - put this on the back burner.  Not sure on your specific applications, but am always interested in saving $$$$ while focusing on quality outcomes.

Bests regards,
John Steele




________________________________
From: Kim Merriam <kmerriam2003 <@t> yahoo.com>
To: "Patten, Nicole (NIH/NIAAA) [F]" <pattennj <@t> mail.nih.gov>; histonet <@t> lists.utsouthwestern.edu
Sent: Wednesday, December 10, 2008 11:05:40 AM
Subject: Re: [Histonet] Primary works with ABC but not IF

Hi Nicole,

I don't know what kind of secondary antibody you are using, but we have had very good luck with the Alexa-Fluor secondaries.  Often, when you go from an amplified chromagenic method (such as ABC) to a less-amplified staining method (such as with a fluorescently-labeled secondary), the primary antibody needs to be retitrated.  Perhaps there is not enough amplification with a directly labeled seconday; you may need to add another lay or do additional amplification, such as with tyramide - we are having very good luck with Invitrogen's TSA kits that contain AlexaFluor dyes.  The Perkin Elmer FITC-TSA kits also work well, but cost about 3X as much as Invitrogens.

If you send more information about your staining protocols, we could probably give you more specific advice.

Kim
 Kim Merriam, MA, HT(ASCP)QIHC
Cambridge, MA 




________________________________
From: "Patten, Nicole (NIH/NIAAA) [F]" <pattennj <@t> mail.nih.gov>
To: histonet <@t> lists.utsouthwestern.edu
Sent: Wednesday, December 10, 2008 10:09:45 AM
Subject: [Histonet] Primary works with ABC but not IF

Hello-

I am in a situation where my primary antibody works using the ABC
method, but I do not see staining with immunofluorescence. My IF
protocol works well with other antibodies so I do not know why it's not
working with this particular antibody.. Does anybody have any
suggestions?

I have considered using an avidin-Alexa Fluor conjugate but I am not
sure if this is even possible (I am new to IHC) or how I would go about
it. Would I need to first biotinylate my primary?

Does anyone have any advice/protocols? 

Thanks! Any help would be greatly appreciated.

Nicole Patten
Post-Baccalaureate Fellow/IRTA
NIAAA/National Institutes of Health


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