Re: FluoroNanogold background in the mitochondria **vendor reply**

[Kilgore, Jason A.]

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** Vendor Reply **

Hi, Sean,

One possibility is that the negatively-charged Alexa Fluor dye is attracted to the positively-charged mitochondria.  This dye-based background binding isn't blocked by protein blocking, such as with non-fat milk.

There is a blocker for such charge-based binding, called the Image-iT FX Signal Enhancer solution, which is sold by Thermo Fisher as catalog number I36933 or R37107.  After fix and perm, apply the ready-made, 1X solution for 20 minutes, then wash off and proceed with the rest of the usual protocol.

Cheers,

Jason

Jason A. Kilgore
Technical Application Scientist
Molecular Probes Tech Support
Life Sciences Solutions

Thermo Fisher Scientific
29851 Willow Creek Rd.
Eugene, OR  97402-9132
1-800-955-6288 then option 4, then option 6, then option 2.
Or dial direct at [phone]
[hidden email]
www.lifetechnologies.com

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sean Speese
Sent: Friday, February 12, 2016 11:28 AM
To: [hidden email]
Subject: FluoroNanogold background in the mitochondria

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We are trying a new pre-embed immunoEM protocol using streptavadin-Alexa 546/1.4nm nanogold conjugate (Nanoprobes) as our tertiary detection reagent. We are using the probe in fixed Drosophila body wall muscle, and we are getting a lot of signal from the mitochondria, however our primary antibody very cleanly stains the nuclear lamina. It seems that the nanogold is the culprit, and we are not sure how to effectively block this cross reaction.  We have used 5% non-fat milk and that has helped, but still leaves substantial background in the mitochondria. Just wondering if anyone else has come across this problem.  

[Konstantín Levitskiy]

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Hi Jason.

As I remember the mitochondria inner side is very negatively charged, is
about -200 mV, compared to an inside of the cytoplasm (-80mV) with regard to
outside. And the charge is losing during the cell fixation.

Best regards,
Dr. Konstantín Levitskiy
Servicio de Microscopía
InstitutodeBiomedicinadeSevilla - IBiS
Campus del Hospital Universitario Virgen del Rocío
Avda. Manuel Siurot s/nº
41013 Sevilla
Tlfno: 955 92 3030
Email: [hidden email]
Web: www.ibis-sevilla.es

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-----Mensaje original-----
De: Confocal Microscopy List [mailto:[hidden email]] En
nombre de Kilgore, Jason
Enviado el: lunes, 15 de febrero de 2016 20:53
Para: [hidden email]
Asunto: Re: FluoroNanogold background in the mitochondria **vendor reply**

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** Vendor Reply **

Hi, Sean,

One possibility is that the negatively-charged Alexa Fluor dye is attracted
to the positively-charged mitochondria.  This dye-based background binding
isn't blocked by protein blocking, such as with non-fat milk.

There is a blocker for such charge-based binding, called the Image-iT FX
Signal Enhancer solution, which is sold by Thermo Fisher as catalog number
I36933 or R37107.  After fix and perm, apply the ready-made, 1X solution for
20 minutes, then wash off and proceed with the rest of the usual protocol.

Cheers,

Jason

Jason A. Kilgore
Technical Application Scientist
Molecular Probes Tech Support
Life Sciences Solutions

Thermo Fisher Scientific
29851 Willow Creek Rd.
Eugene, OR  97402-9132
1-800-955-6288 then option 4, then option 6, then option 2.
Or dial direct at [phone]
[hidden email]
www.lifetechnologies.com

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Sean Speese
Sent: Friday, February 12, 2016 11:28 AM
To: [hidden email]
Subject: FluoroNanogold background in the mitochondria

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

We are trying a new pre-embed immunoEM protocol using streptavadin-Alexa
546/1.4nm nanogold conjugate (Nanoprobes) as our tertiary detection reagent.
We are using the probe in fixed Drosophila body wall muscle, and we are
getting a lot of signal from the mitochondria, however our primary antibody
very cleanly stains the nuclear lamina. It seems that the nanogold is the
culprit, and we are not sure how to effectively block this cross reaction.
We have used 5% non-fat milk and that has helped, but still leaves
substantial background in the mitochondria. Just wondering if anyone else
has come across this problem.


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