Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
A few additional suggestions:
Without removing resin probes will indeed only bind to the
surface and in general fluorescent labelling is likely to be
invisible but colloidal gold with silver enhancement will
work if you have a high-contrast imaging system such as
reflection confocal or darkfield.
Epon will do better than Spurrs - it's a bit more permeable to
water. But why not use LR White or Gold which will work fine
and are also OK for EM? They are also very low viscosity
(unlike EPON).
If you have osmium in your tissue it will have fairly
unfortunate effects - it can in principle be removed
with hydrogen peroxide but it's better just to avoid it.
If you must use Spurrs then etch it down - I've always
used sodium methoxide (drop bits of sodium in methanol -
great fun!). That may be what the commercial kit is - I
don't know. Either way handle these reagents with very
great care!
If you have dried down your sections on to coverslips
they will stay put with no problems during these procedures -
that is the least of your problems.
Guy
Diana van Driel wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> I suggest you try the Microscopy list. I'm sure someone there will have
> an answer.
> The address is http://microscopy.com/MicroscopyListserver/
>
> In general, trying to label epoxy is unrewarding - it's very
> impermeable, but removing it is damaging. Specifically, for your
> problem, I can't help.
>
> Cheers,
>
> Diana
>
> Diana van Driel
> Dept Ophthalmology
> Sydney University
> GPO Box 4337
> Sydney, NSW
> AUSTRALIA 2001
>
> Phone 61 2 93827278
> Mobile 0423 151614
> FAX 61 2 93827318
> On 31 Aug 2005, at 6:59 AM, Sheri Simmons wrote:
>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Hi,
>>
>> I'm hoping someone on the list has some expertise that they could share
>> with me. I've searched the list archives but didn't turn up anything. I'm
>> working with some thick sections (about 1 mm) embedded in Spurr's low
>> viscosity epoxy resin. The goal is to incubate these sections with
>> fluorescently labelled nucleic acid probes. The first thing I plan to do
>> is try incubating these with a Polysciences epoxy removal kit to make the
>> cells accessible for staining. I'm wondering if anyone has experience
>> with
>> using nucleic acid probes on thin sections, or could direct me to some
>> good references. Is there anything I could use to increase the
>> permeability of the epoxy, or are my probes just going to bind to
>> whatever
>> is exposed with the epoxy removal solution?
>> Also, could anyone recommend a way to fix the sections on coverslips for
>> manipulation in the staining procedure and later viewing with LSCM?
>>
>> Thanks,
>> Sheri
>>
>>
>> ---------------------------------------------------
>> Sheri Simmons
>> Geomicrobiology Group
>> MIT-WHOI Joint Program, Biological Oceanography
>> http://www.whoi.edu/science/MCG/edwards/sherisimmons
>>
--
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone +61 2 9351 3176 Fax +61 2 9351 7682
Mobile 0413 281 861
______________________________________________
http://www.guycox.net
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