RE: clip-on in situ hybridization chambers
|From:||"Su, Phy-Huynh" <firstname.lastname@example.org>|
I have used those Probe-on Chambers for years, and loved them. They come in
various sizes: 50ul, 100ul and 200ul (20x40mm).
They saved me precious reagents, and are quite sealed. However, I wouldn't
recommend to re-use them for in-situ hybridization because of chances of
RNAse contamination (the new ones are DNAse and RNAse free), and they loose
their adhesiveness after being washed (the rubber like structures will get
detached from the coverslip) thus will not seal well.
It's OK to use for Immunohistochemistry because the temperatures being used
are not as hi, and we don't have to worry about RNAse problems. I've washed
them with mild detergents for re-use.
Make sure to use their recommended volumes.
To take the chambers out, immerse them in a beaker filled with washing
buffer. Use a pair of forceps, and gently pry at one corner to let fluid
inside the chamber. Be patient, and let the buffer wash off the chamber for
you. But use the forceps to guide the chamber away from your tissues,
otherwise it can scratch them off.
My humble experience.
> -----Original Message-----
> From: Karin Pittman [SMTP:email@example.com]
> Sent: Thursday, February 08, 2001 5:03 AM
> To: HistoNet Server
> Subject: Re: clip-on in situ hybridization chambers
> Dear Histonetters
> I realize I may have been unclear in describing our routines for in situ
> hybridizations, and the reason for my plea for help. We routinely use
> coverslips or even parafilm to keep the moisture in on the slides, but are
> working with very thin sections which are often destroyed during the
> process of removing the coverslips. We had bought a special chamber for in
> situ incubations (a Grant Boekel SM 30) to try to avoid using coverslips
> but, despite various forms of keeping the conditions stable throughout,
> humidity high and even covering the cover to eliminate a temperature
> differential, what we get are large drops of condensation on the upper
> surface of the chamber which drip down again onto the slides (which have
> the meantime dried out slightly) causing wonderful patterns of artifacts
> and no reliable results. Using the coverslips inside the chamber gets us
> back to step 1 again and loss of tissue.
> Then we recently noticed that Sigma produces "Probe-clip press-seal
> incubation chambers" (catalogue number Z35,947-5) which are 22 x 40 mm for
> clipping right onto the slide. They are in effect microchambers, covering
> but not touching the tissue. This in theory would be fine if they too dont
> dry out. They sell in packages of 25 and are re-useable. Now my original
> question: Does anyone have any similar experience and has anyone tried
> these microchambers?
> thanks for your help
> Dr. Karin Pittman
> Assoc. Professor
> Dept. of Fisheries and Marine Biology
> University of Bergen
> 5020 Bergen
> tel: 47 55 58 44 72 (direct)
> fax: 47 55 58 44 50
> email: firstname.lastname@example.org
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