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It is not correct to say that ProLong Gold outperforms Vectasheild with
alexa dyes. Invitrogen's own data show that Vectasheild outperforms
ProLong Gold with Alexa dyes excitable at wavelengths of 488 nm and
less. It is only the Alexa dyes that are excitable in the green and
particularly the red part of the spectrum that significantly benefit
from ProLong.
Simon
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Mariette P.C. Kemner - van de Corput
Sent: 06 March 2008 12:21
To: [log in to unmask]
Subject: Re: Secondary antibody for 405nm laser line ?
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For those not knowing, alexa fluor dyes are reported to bleach more
quickly when embedded in vectashield. Invitrogen has ProLong (gold)
antifading embedding medium that is more suitable for all alexa dyes.
The
bleaching is much less and the dyes are more stable over time. I have
re-used slides that were almost 1 year old that were stored at 4oC. The
dyes were less bright and bleached more quickly but for a year old slide
I
was surprised it was still that good.
Pro-long gold needs curing overnight and at least 4 days of absolute
curing. Only then the refractive index gets to 1.45. It is not wise to
immediatly go look at the slides when just embedded. The signals will be
weak and blurry.
As for blue dyes. My experience with Alexa Fluor 350 is not that good.
It
works but for small targets the dyes are too weak as compared to
Alexa488
or 594. For bigger/abundant targets it's fine.
Mariette
Mariette
____________________________________________
Dr. M.P.C. Kemner-van de Corput
____________________________________________
MGC - Dept. of Cell Biology & Genetics
Erasmus Medical Center
Dr. Molewaterplein 50, 3015 GE Rotterdam
POB 2040, 3000 CA Rotterdam, The Netherlands
Delivery adres:
Erasmus MC, Westzeedijk 353, 3015 AA Rotterdam,
The Netherlands
Office: H-Ee751; tel: +31 10 704.3949
Lab: H-Ee710; tel lab: +31 10 704.3315
tel secr: +31 10 704.3169
____________________________________________
http://www2.eur.nl/fgg/ch1/cellbiology/
http://www.thesis.kemner.biz/
____________________________________________
Op Wo, 5 maart, 2008 5:17 pm, schreef simon walker (BI):
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> We have used Alexa Fluor 405 (Invitrogen) with limited success. Our
> experience of it is that it's reasonably bright, but bleaches very
> quickly both under epi-illumination and with confocal lsm. Mounting
> with an antifade seems to help (we've used Vectasheild), but it still
> bleaches very quickly relative to, for example, AF488. If someone has
> found a bright and stable 405-excitable fluorescent dye I would be
very
> interested to know. Perhaps Qdots maybe a possible alternative?
>
> Simon
>
> Babraham Institute
>
> Cambridge
>
> UK
>
>
>
> ________________________________
>
> From: Confocal Microscopy List [mailto:[log in to unmask]]
On
> Behalf Of Christophe Leterrier
> Sent: 05 March 2008 15:46
> To: [log in to unmask]
> Subject: Secondary antibody for 405nm laser line ?
>
>
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear list,
>
> I would like to use all four lines of our new confocal for
> immunolabeling (405,488,532,633). For the moment I use
> Hoechst/Alexa488/Alexa555/Cy5, but I would like to have a fourth
> antibody labeling using the 405 channel instead of Hoechst. What
> secondary fluorescent antibody would you recommend ? Is there an
> Alexa405 or something similar that would be bright and stable ?
>
> Thanks a lot,
>
> Christophe Leterrier
>
> Postdoc
> INSERM UMR641 Neurobiology of ionic channels
> IFR Jean Roche - Mediterranee University
> Marseille, France
>
>
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