Dear Keith and Carol,
Thanks for the kind advice. And perhaps I should elaborate our
experiment conditions a bit more.
We are using two high NA lens from different vendors, both for TIRF
experiment.And to match the high NA of the lens, the coverslips need to
have high RI, hence are not made of normal glass( at least not
entirely). One of the lens is not commercialized yet, neither does the
coverslips for the lens, so we don't know what's in there exactly. The
other one is the 1.65 NA lens from Olympus, and the coverslip should be
made of sapphire. As measured by micrometer, the thickness is around 0.17mm.
As for the sample preparation, we wash the coverslips as normal(10M
nitric acid, milli Q washing, air dry), coat them with fibronectin and
grow cells on them. They are used for TIRF imaging, hence no mounting
media but medium with matching RI with water(PBS/water) on top of them.
Hence removing mounting media is not really our concern, we are more
nervous about getting cells off as well as the fibronectin. Once these
are removed, we will probably use the old method again to clean the
coverslip.
Thanks for helping.
Regards,
Edna, HU Xian
Keith Morris wrote:
> Likewise:
>
> We remove the coverslip by soaking in 2X SSC [probably for no real reason,
> but it helps preserve the specimen on the slide, and I suppose you could add
> a drop of Tween 20 to the Coplin jar]. Recover and then rinse the coverslip
> in deionised water, and finally wipe the coverslip carefully with ether or
> petroleum spirit in the fume cupboard [gloves + KimTech Science 75512
> tissues] to get it as clean as possible before the wash sequence below. We
> use immersion oil for imaging [which doesn't dissolve so well in ethanol,
> hence ether/petroleum spirit], and more importantly 'always liquid'
> VectaShield + DAPI non-hardening mountant [naturally with no nail polish
> gluing it on]. You can generally slowly soak off 'permanently' mounted
> coverslips with things like Xylene/Toluene [check what the original mountant
> was dissolved in, e.g. Histomount uses toluene]. After ether/petroleum
> spirit cleaning, quickly visually inspect the cover-slips at an angle to the
> reflected light to ensure they are smear free before the wash sequence
> below.
>
> [This wash sequence is also used for new 'pre-washed' glass slides from
> boxes]: Soak the cleaned coverslips overnight in 5ml Teepol/RenClean in ~500
> ml [try a slotted rack to hold the coverslips upright]. Wash off detergent
> gently with tap water then de-ionised. Leave for 1h in ~500 ml de-ionised +
> 5ml conc HCL. Rinse with tap water then de-ionised, and put in 100% ethanol
> for 1h. Replace with fresh 100% ethanol for another hour. Remove Ethanol
> and air dry in covered chamber. We generally save the last ethanol wash to
> re-use as the next 'first' one. I always wipe the cover-slip again with 70%
> ethanol and KimTech Science 75512 tissues [wet then dry side], and leave to
> dry just before use or reuse.
>
> Haven't need to do this for a while, but it used to work. It's expensive in
> time and materials though, and you ideally need some sort of rack for the
> washes [easy if you have an in-house workshop].
>
> Keith
> ---------------------------------------------------------------------------
> Dr Keith J. Morris,
> Molecular Cytogenetics and Microscopy Core,
> Laboratory 00/069 and 00/070,
> The Wellcome Trust Centre for Human Genetics,
> Roosevelt Drive,
> Oxford OX3 7BN,
> United Kingdom.
>
> Telephone: +44 (0)1865 287568
> Email: [log in to unmask]
> Web-pages: http://www.well.ox.ac.uk/cytogenetics/
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]] On
> Behalf Of Hu Xian
> Sent: 18 May 2009 07:55
> To: [log in to unmask]
> Subject: Protocols for reusing coverslips
>
> Dear List,
>
> We need to use some pretty expensive coverslips for high NA
> objectives(around 8 USD per piece). We might have to reuse them :(.
> Any one has any established protocol for coverslip re-use for share?
> Suggestions are welcomed too.
>
> Thanks a lot...
>
>
> Regards,
> Edna
>
>
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