Kilgore, Jason A. |
*****
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. ***** ** 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 +1 541 335 0353 [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. |
Konstantín Levitskiy |
*****
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. ***** 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 AVISO SOBRE CONFIDENCIALIDAD. Este mensaje y sus anexos son confidenciales y pueden estar protegidos por disposiciones legales. Si Vd. no es el destinatario del mismo, por favor, notifíquenoslo inmediatamente y destruya o devuelva el original. No deberá copiar este mensaje ni sus anexos o usarlo para propósito alguno, ni divulgar su contenido a ninguna persona. P El medio ambiente es nuestra responsabilidad. Por favor, antes de imprimir este mensaje, compruebe que sea necesario hacerlo. -----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** ***** 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. ***** ** 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 +1 541 335 0353 [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. --- El software de antivirus Avast ha analizado este correo electrónico en busca de virus. https://www.avast.com/antivirus |
Free forum by Nabble | Edit this page |