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I agree.  Lose the Vectashield.  Switching to Prolong Gold will allow  
you to preserve your signal for months.  None of the mounting medias  
for fluorescence will completely solve your RI mismatch.  Prolong,  
for example, goes from 1.39 to 1.45 as it dries (see the Invitrogen  
spec sheet), but you will never reach 1.518 (oil).  Mowiol is a bit  
closer (1.49), and Pawley says that 84% sucrose is the same as oil.   
Has anybody successfully implemented 84% sucrose as a fluorescent  
mounting medium?

No commercial affiliation.

Michael J. Schell,
Assist. Professor
Dept. of Pharmacology
Uniformed Services University
4301 Jones Bridge Rd.
Bethesda, MD  20814-3220
tel:  (301) 295-3249
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On Oct 20, 2007, at 12:26 PM, Connell, Samuel wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> It sounds like using Prolong Gold from Molecular Probes  
> (Invitrogen) would make the most sense in your situation.  If used  
> correctly, it would certainly help with the Alexa 488 signal and  
> provides a RI that should be matched to your immersion oil.
>
> No commercial affiliation.
>
> --
> Samuel A. Connell
> Director, Light Microscopy
> Cell & Tissue Imaging Center
> St. Jude Children's Research Hospital
> 332 North Lauderdale St., E7061
> Memphis, TN 38105-2794
> (901) 495-2536
> [log in to unmask]
>
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List on behalf of Farid Jalali
> Sent: Sat 10/20/2007 10:22 AM
> To: [log in to unmask]
> Subject: Alexa488 staining question.    .
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hello All,
> This is a question about using Molecualr Probes Alexa 488 Donkey  
> Anti-Mouse
> secondary antibodies.
> I am using this antidody to detect a number of different targets  
> and have
> noticed that the signal intensity fades over the course of 7-10 days.
> I am using it for indirect immunofluorescent detection of targets  
> that form
> intra-nuclear foci ranging in size from ~300nm to 2um. Compared to
> the distinct, punctate, bright
> foci I image within 1-2 days after staining, 7-10 days later they are
> smaller, more diffuse and not bright at all. Using the same  
> exposure times
> and gain setting does not yeild the same quality of images.
> This is an obvious problem as I am trying to be consistent with my  
> settings
> from sample to sample for quantitation.
>
> Has anyone come across a problem like this previously? I am  
> mounting my
> coverslips with Vectashield. Has anyone come across an antifade  
> mountant
> that has an RI closer to that of immersion oil?
>
> Thanks to all.
> Cheers
> Farid
>
> -- 
> Farid Jalali MSc
> Senior Research Technician/ Lab Manager
> Dr. Robert Bristow Lab
> Applied Molecular Oncology
> Princess Margaret Hospital
> Toronto, Canada
> 416-946-4501 X4351 (Princess Margaret Hospital)
> 416-581-7754 STTARR at MaRS Building
> 416-581-7791 STTARR Microscopy Suite