how to measure acidification in endosomes?
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Rietdorf, Jens |
how to measure acidification in endosomes?
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Dear colleagues,
to demonstrate a receptor has indeed been internalized (and he is not just looking at a structure still continuous with the plasma membrane), a user of our facilities wants to measure the acidification of the endosome compartment (its co-localized with EEA1). Which is a good protocol to do so? Which alternative strategy would be applicable? Any suggestion welcome, Thanks, jens --- dr. jens rietdorf head microscopy novartis research foundation friedrich-miescher-institute, wro1066.2.16 maulbeerstr.66, 4058 basel, switzerland couriel:rietdorf(at)fmi(dot)ch fon: +41.61.69.75172 fax: +41.61.69.73976 |
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Dries Vercauteren |
Re: how to measure acidification in endosomes?
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Dear Dr Rietdorf and others,
indeed, also in our research we are very much interested in measuring endosomal pH. One strategy, I was thinking, was using a pH sensitive dye, with a precise defined pKa value like Lysosensor green (LysoSensor™ Green DND-189 pKa 5.2; LysoSensor™ Green DND-153 pKA 7.5), or use SNARF-1, conjugated to a low MW dextran, in that way you get a pH shift when the endosome is acidifying (pKa 7,5). According to Mol Probes (http://probes.invitrogen.com/media/pis/mp01270.pdf) it should even be possible to calculate the pH after calibrating and solely measuring emission intensities at two different wavelenghts.. However, I didn't do this myself yet.. LysoSensor™ Green DND-189 pKa 5.2; LysoSensor™ Green DND-153 pKA 7.5 Another possibility is measuring fluorescence of simultaneously added FITC and TMR: Sonawane, N. D., Szoka, F. C., and Verkman, A. S. (2003). Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes. Journal of Biological Chemistry. 278: 44826-44831. Anyone who has experience with this or other suggestions are all wellcome ! Kind regards, Dries. 2009/5/15 Rietdorf, Jens <[hidden email]> Dear colleagues, -- Dries Vercauteren, PhD student Master of Bioscience Engineering: Cell and Gene Biotechnology Ghent Research Group on Nanomedicines www.ugent.be/fw/en/research/biofys Faculty of pharmaceutical sciences, Ghent University Harelbekestraat 72, 9000 Ghent Belgium Phone: +329/264 80 49 Mobile: +32485/30 69 80 E-mail: [hidden email] [hidden email] |
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"José A. Feijó" |
Re: how to measure acidification in endosomes?
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if it is a transformable system, I believe pHluorins may be where you
want to go, check the original paper by Miesenboeck and Rohtman:
Miesenbock, G., De Angelis, D. A. & Rothman, J. E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998). I believe you can get the construct directly from the Mount Sinai lab, with the endocytic receptor and a mammalian promotor, thus directly usable, in 3 different flavours according to your imaging system/application Dries Vercauteren escreveu: Dear Dr Rietdorf and others, -- ********************************************************** Jose' A. Feijo', Prof. ---------------------------------------------------------- Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa PT-1749-016 Lisboa, PORTUGAL tel. +351.21.750.00.47/00/24, fax +351.21.750.00.48 and/ e Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70 __________________________________________________________ e.mail: [hidden email] URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38 ********************************************************** |
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Ignatius, Mike |
Re: how to measure acidification in endosomes? Response from Molecular Probes
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Dear Dr Rietdorf
Along with the suggestions already
made, I would recommend reading our literature on the latest addition to the
selection of pH sensitive dyes, pHrodo, succinimidyl ester (pHrodo, SE)
SKU#
Mike Ignatius From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of "José A. Feijó" Sent: Friday, May 15, 2009 6:15 AM To: [hidden email] Subject: Re: how to measure acidification in endosomes? Miesenbock, G., De Angelis, D. A. & Rothman, J. E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192195 (1998). I believe you can get the construct directly from the Mount Sinai lab, with the endocytic receptor and a mammalian promotor, thus directly usable, in 3 different flavours according to your imaging system/application Dries Vercauteren escreveu: Dear Dr Rietdorf and others, -- ********************************************************** Jose' A. Feijo', Prof. ---------------------------------------------------------- Dep. Biologia Vegetal, Fac.Ciencias, Universidade Lisboa PT-1749-016 Lisboa, PORTUGAL tel. +351.21.750.00.47/00/24, fax +351.21.750.00.48 and/ e Inst.Gulbenkian Ciencia, PT-2780-156 Oeiras, PORTUGAL tel. +351.21.440.79.41/00/19, fax +351.21.440.79.70 __________________________________________________________ e.mail: [hidden email] URL: http://www.igc.gulbenkian.pt/code/research.php?lang=en&unit_id=38 ********************************************************** |
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Rosemary.White |
Re: how to measure acidification in endosomes?
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In reply to this post by "José A. Feijó"
cheers, Rosemray Rosemary White CSIRO Plant Industry GPO Box 1600 Canberra, ACT 2601 Australia ph 61 2 6246 5475 fx 61 2 6246 5334 On 15/05/09 11:14 PM, "José A. Feijó" <jfeijo@...> wrote: if it is a transformable system, I believe pHluorins may be where you want to go, check the original paper by Miesenboeck and Rohtman: |
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In reply to this post by Dries Vercauteren
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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Carol Heckman |
Re: Protocols for reusing coverslips
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Edna-
We don't exactly re-use them, but we do clean them for hours (12 h) in 1 N HCl and then wash exhaustively in deionized water, followed by several rinses in 95% ethanol and air-drying. This takes off everything left by the manufacturing process and leaves a molecularly clean glass surface. If you actually grew cells on the coverslips, it might not be drastic enough to clean off the cell debris, though. Carol Heckman Center for Microscopy & Microanalysis Bowling Green State University ________________________________________ From: Confocal Microscopy List [[hidden email]] On Behalf Of Hu Xian [[hidden email]] Sent: Monday, May 18, 2009 2:54 AM To: [hidden email] 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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Keith Morris |
Re: Protocols for reusing coverslips
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In reply to this post by Hu Xian-3
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: [hidden email] Web-pages: http://www.well.ox.ac.uk/cytogenetics/ -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Hu Xian Sent: 18 May 2009 07:55 To: [hidden email] 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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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: [hidden email] > Web-pages: http://www.well.ox.ac.uk/cytogenetics/ > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On > Behalf Of Hu Xian > Sent: 18 May 2009 07:55 > To: [hidden email] > 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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Keith Morris |
Re: Protocols for reusing coverslips
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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: [hidden email] Web-pages: http://www.well.ox.ac.uk/cytogenetics/ -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Hu Xian Sent: 19 May 2009 05:07 To: [hidden email] 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: [hidden email] > Web-pages: http://www.well.ox.ac.uk/cytogenetics/ > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] > Behalf Of Hu Xian > Sent: 18 May 2009 07:55 > To: [hidden email] > 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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In reply to this post by Hu Xian-3
We have the same cover glass for TIRF, even thoough we have not tried
this on these slips we clean all our lithographic glass substrates with piranha at a 4:1 ratio. http://en.wikipedia.org/wiki/Piranha_solution It eats off almost every organic material- leave it in for 10min and rinse a bunch with DI water. We do this is a hood and waste it in a special container with a vented cap. It is very nasty and exothermic so only make a small amount in a beaker. Try one Si slip in this and see what happens- actually let me know. I am not sure the chemistry of the shaphire is exactly the same as glass but it should not be that different for short term exposures. best Julia Sheetz lab On May 19, 2009, at 12:07 AM, Hu Xian wrote: > 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: [hidden email] >> Web-pages: http://www.well.ox.ac.uk/cytogenetics/ >> -----Original Message----- >> From: Confocal Microscopy List >> [mailto:[hidden email]] On >> Behalf Of Hu Xian >> Sent: 18 May 2009 07:55 >> To: [hidden email] >> 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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Keith Morris |
Re: Protocols for reusing coverslips
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Yep it's basically hydrogen peroxide and a strong acid. Chemicals of these
sort have been used for lung tissue digestion* for many decades [perhaps most are not made quite as aggressive as 'Piranha solution' though]. Tissue digestion protocols are typically based on strong acids [hydrochloric, glacial acetic], hydrogen peroxide, bleach [5% sodium hypochlorite], formamide, plasma ashing and proteolytic enzymes - all to typically to digest away lung tissue and to recover inhaled particulate matter**. Sometimes it's a combination of these chemicals/procedures. The method used is solely selected in order to minimise damage to the delicate material that you want to recover, e.g. inhaled coal particles, glassfibres, asbestos, talc, mineral dust, paramid fibres, diatoms** etc.... If the inhaled material was say bleach sensitive, then an enzymatic digestion would be used. Provided your cover-slip is completely resistant to these chemicals, the use of any of the above should provide a successful digest and removal of the cells and fibronectin. Piranha solution sounds a pretty aggressive tissue digestant though, 'making the glass hydrophilic by hydroxylating the surface' - and for disposal we could just pour our bleach down the drain. Keith *unlike lung, fatty tissues are far harder to digest. ** Sometimes to check for sea drowning, you look for the presence of diatoms in the lung --------------------------------------------------------------------------- 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: [hidden email] Web-pages: http://www.well.ox.ac.uk/cytogenetics/ -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of js1719 Sent: 20 May 2009 03:03 To: [hidden email] Subject: Re: Protocols for reusing coverslips We have the same cover glass for TIRF, even thoough we have not tried this on these slips we clean all our lithographic glass substrates with piranha at a 4:1 ratio. http://en.wikipedia.org/wiki/Piranha_solution It eats off almost every organic material- leave it in for 10min and rinse a bunch with DI water. We do this is a hood and waste it in a special container with a vented cap. It is very nasty and exothermic so only make a small amount in a beaker. Try one Si slip in this and see what happens- actually let me know. I am not sure the chemistry of the shaphire is exactly the same as glass but it should not be that different for short term exposures. best Julia Sheetz lab On May 19, 2009, at 12:07 AM, Hu Xian wrote: > 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: [hidden email] >> Web-pages: http://www.well.ox.ac.uk/cytogenetics/ >> -----Original Message----- >> From: Confocal Microscopy List >> [mailto:[hidden email]] On >> Behalf Of Hu Xian >> Sent: 18 May 2009 07:55 >> To: [hidden email] >> 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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