Hi Edna,
I though it might be TIRF coverslips [obviously not Sapphire ones from the
$10 cost].
Back in my inhalation fibre toxicity/bio-durability days I recovered the
inhaled glassfibres from the lung tissue using bleach. The lungs were always
digested in bleach for 1 to 4 hours, in a large centrifuge tube on a
motorised roller at 4oC in a cold room [probably in 50ml bleach]. You nipped
in every few hours and the lungs would gradually get smaller and smaller and
then disappear from view [fully digested]. Your cells adhered to coverslips
naturally won't need long. The bleach completely liquidises the lung tissue
such that the tissue digest fully passed through submicron pore size
Nucleopore filters, leaving just the recovered glass fibres on the filter
surface for viewing via SEM or optical microscope using standard filter
optical clearing techniques [making the filter transparent].
Well actually it was 14% hypochlorite solution we used latterly to digest
the tissues, although traditionally household bleach was used - always the
cheapest stuff, not the one with thickeners, and this would be a tad milder
and have detergents added.
Bleach didn't affect the glass fibres and from EDX SEM elemental analysis
measurements, chlorine was only found within very eroded glass fibres - MMVF
fibres tend to dissolve faster in the mildly alkaline lung surfactant
[glassfibres] or acidic alveolar macrophage phagolysosomes [Rockwool]
depending on fibre composition. Unlike other tissue digestion methods [e.g.
plasma ashing], bleach at 4oC was found to minimise fibre breakage during
recovery [eroded micron diameter glass fibres can be very very fragile after
months/years in the lung]. Thus bleach should be glass friendly, easily
washed off, and OK for use with your coverslips. I would think around 10 or
so minutes in bleach would be enough for cells on a coverslip, maybe longer
or less even at RT or say 37oC. I wouldn't have thought the fibronectin
would be a particular problem either - but try it and see.
Once the cells have been digested off the coverlips, you can try the rest of
the cleaning protocol I mentioned [including perhaps a detergent pre-wash
and then ether/petroleum spirit manually cleaning] after the 'digested'
slides have been washed off in de-ionised water. I see no reason why bleach
shouldn't be work well, although you'll have to adapt the digestion
agitation system and a rack would again be useful. The only proviso is that
you wouldn't want any chlorine remaining on the glass surface when
re-culturing [not something I've tried] - but we saw no evidence of that on
our intact [non-eroded] glassfibres' with SEM elemental analysis.
Others have used enzymes or even sodium hydroxide and plasma ashing to
recover fibres from lungs and 'digest'/remove the cellular material:
e.g. http://annhyg.oxfordjournals.org/cgi/reprint/41/6/721.pdf
The alternative enzyme digestion might appeal, but I suppose enzymes might
stick to glass more than bleach and might be a bit specific rather than a
general digest [or they might not].
Regards
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: 19 May 2009 05:07
To: [log in to unmask]
Subject: Re: Protocols for reusing coverslips
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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