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Author Topic: non-specific binding of Alexa-488  (Read 7220 times)

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Offline Raj vasireddy

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non-specific binding of Alexa-488
« on: January 14, 2009, 08:34:46 PM »
Dear all,
Happy New year. I am doing triple labeling of my cells (Alexa 488, 555 and TOPRO 3) and observed that Alexa 488 (but not 555 and TOPRO3) is giving a lot of background noise on my confocal imaging (Its sticking on slide a lot). I am using both the secondaries at 1: 500 dilution (in 1% BSA). I block my cells (in 1% BSA) for 3X 10 mins before adding primary Ab's and incubate slides overnight at 4 degress after adding primary antobodies and then wash with PBS and add secondary Ab's. I use 0.1% TritonX- 100 for permeabilisation. I am using lymphoblastoid cells and I cytospin them for staining. I fix my cells in 4% PFA at RT for 10 mins. I am looking for nuclear antigens. Guys any suggestions about how can I get rid of 488 stickiness.

Thanks heaps
Raj.
« Last Edit: January 14, 2009, 08:52:23 PM by Raj vasireddy »

non-specific binding of Alexa-488
« on: January 14, 2009, 08:34:46 PM »

Offline excalibur

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Re: non-specific binding of Alexa-488
« Reply #1 on: January 17, 2009, 11:33:18 AM »
If you are using Plus slides, I have found it helps to use the blocking on the entire slide. The protein will stick to the slides, but will not stain.

Plus slides can grab in places where there is no tissue.
Paula Keene Pierce, HTL(ASCP)HT
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Offline richard03

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Re: non-specific binding of Alexa-488
« Reply #2 on: January 17, 2009, 02:25:03 PM »
The background could be autofluorescence. Check the slides before staining to make sure this is not the case.

Offline ImmunoNYC

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Re: non-specific binding of Alexa-488
« Reply #3 on: January 19, 2009, 04:01:04 PM »
Some thoughts ...

Are your antibodies fresh? Are they from Molecular Probes? Did you truy diluting it out further? Are you getting any specific signal? Have you tried to increase your blocking with BSA or serum from the host animal? Is your BSA globulin free?

Using confocal stickiness on the slide shouldn't play a major role in imaging as you shouldn't have to image the glass, even if z-stacking.

Re: non-specific binding of Alexa-488
« Reply #3 on: January 19, 2009, 04:01:04 PM »