mounting medium for TIRF
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Dear all,
I'd like to mount fixed cells on a coverslip to observe them by TIRF. Usually I use Mowiol to mount fixed cells but I realized that the refractive index gap necessary for TIRF between the glass and the medium is not present in Mowiol. What medium with a refraction index similar to water can I use ? Is PBS/Glycerol OK for TIRF ? On a related note, I hate nail polish, how can i seal PBS/gly mounted coverslips ? Thank you for your advices Christophe Leterrier |
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Sealing your coverslip can be accomplished with molten
household wax, heated in a beaker or paraffin heater and applied with a Qtip
applicator, or use molten VALAP.
Cheers,
Jason From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier Sent: Friday, February 08, 2008 8:30 AM To: [hidden email] Subject: mounting medium for TIRF I'd like to mount fixed cells on a coverslip to observe them by TIRF. Usually I use Mowiol to mount fixed cells but I realized that the refractive index gap necessary for TIRF between the glass and the medium is not present in Mowiol. What medium with a refraction index similar to water can I use ? Is PBS/Glycerol OK for TIRF ? On a related note, I hate nail polish, how can i seal PBS/gly mounted coverslips ? Thank you for your advices Christophe Leterrier |
 
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Boswell, Carl A - (cboswell) |
Re: mounting medium for TIRF
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In reply to this post by lechristophe
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Zeiss has a nice, thought expensive, oil with water
RI. An alternative to nail polish is Permount, the stuff histological
preparations are mounted with. It can be diluted some with xylene if
necessary, is clearly immiscible in water and forms a very tight moisture
barrier. It smells when fresh, though, so should be dried in a
hood.
Carl
Carl A. Boswell, Ph.D.
Molecular and Cellular Biology University of Arizona 520-954-7053 FAX 520-621-3709
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PBS & glycerol will have too high a refractive index for TIRF. For fixed work we usually just use PBS plus an anti-fade like n-propyl galate (0.1%). I also like VALAP (1:1:1 vaseline:lanolin:parafin) for sealing slides. Use as a warmed liquid, solidifies at room temp. Jennifer On Feb 8, 2008 12:25 PM, Carl Boswell <[hidden email]> wrote:
-- Jennifer Waters, Ph.D. Director, Nikon Imaging Center at Harvard Medical School |
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Thank you for your advices, I'll use the PBS moutning +VALAP sealing
method to mount my coverlips. The question is : how do you keep your
PBS+VALAP slides for say more than one week ?
- At 4°C it will allow growth of contaminants (bacteria, fungi...) relatively rapidly. Is it posssible to add something like sodium azide ? Related question : sodium azide is banned here in France, so do you know another efficient conservative ? I usually keep my fixed cells in 0.1% PFA for up to one or two weeks before immunostaining, but I think PFA will be autofluorescent if I put it in the mounting PBS as a conservative. - If I freeze the slides, will the PBS make cristals and damage the cells ? How many freeze/thaw are possible for immunostained cultured cells without compromizong the cells/killing the fluorescence ? Thanks for your help, Christophe Leterrier On Fri, Feb 8, 2008 at 20:06, Jennifer Waters <[hidden email]> wrote:
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