Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protocols and Methods Forum
Register Search Today's Posts Mark Forums Read

Protocols and Methods Forum Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


autofluorescence - Protocols and Methods Forum

autofluorescence - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.

LinkBack Thread Tools Display Modes
Old 11-11-2009, 09:44 AM
Pipette Filler
Points: 24, Level: 1 Points: 24, Level: 1 Points: 24, Level: 1
Activity: 0% Activity: 0% Activity: 0%
Join Date: Nov 2009
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default autofluorescence

hello everybody
I'm doing a PhD in horse virology and it is more than 4 months that I can not develop an efficient protocol for Immunofluorescence of infected PBMC cells
I am using a monoclonal antibody that binds to a nucleocapsid protein. This product is available on the market
my problem is that this mAb binds aspecifically neutrophils (you can identify them for the morphology of the nucleus ... the really strange thing is that using an irrelevant isotype autofluorescence does not develop!
I tried to use a protocol to limit autofluorescence but was not successful
I do not know what to do, time passes and I always stop at the same point

I hope you can help

Reply With Quote
Old 11-11-2009, 02:29 PM
Warthaug's Avatar
Points: 2,383, Level: 31 Points: 2,383, Level: 31 Points: 2,383, Level: 31
Activity: 0% Activity: 0% Activity: 0%
Join Date: Sep 2009
Location: London, Canada
Posts: 203
Thanks: 1
Thanked 68 Times in 61 Posts
Default Re: autofluorescence

Are you purifying the PBMC's? If not you may want to do so in order to avoid contamination. Neutrophils have a large number of Fc receptors, and those may be binding your antibody. Alternatively, the antibody may be recognising an unrelated epitope on the neutrophils.

There are a couple of options:
1) Stain your neutrophils so you can ID them and then eliminate them later from your analysis. There are various neutrophil-specific antibodies you can use for this task (anti-GR1 for example).

2) Purify your prep to remove the contaminating cells. This is rather easy - plate down your prep on a tissue culture dish for 8hrs or so, then wash. Neutrophils should not stick, and will wash off.

3) Buy, or make, your antibody as a Fab or F(ab)2. This will eliminate Fc receptor binding

4) Pretreat your cells with an isotype antibody from the same species (and isotype) as your primary antibody. This should block any Fc interactions, but may activate your cells.

5) Block your cells with serum from the same (or closely related, if same is not available) species as your primary antibody. This should reduce any non-specific binding.

6) Try another antibody, if one is available. Ideally another clone, but even the same clone from a different company can behave differently.

[Only registered users see links. ], here at Molecular Station.
Reply With Quote


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Autofluorescence 123 Zebrafish Forum 0 04-01-2008 07:02 PM
Thiamine autofluorescence ??? Isabelle Jourdain Yeast Forum 0 09-12-2005 05:00 AM
How to avoid autofluorescence when dissect the brian? Haojiang Drosophila Forum 0 10-29-2003 02:35 PM

All times are GMT. The time now is 09:24 AM.

Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2015, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.12092 seconds with 16 queries