RE: IHC mounting media
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|From:||Paul Klosen <firstname.lastname@example.org>|
|To:||"Nocito, Joseph" <email@example.com>, "'P. Emry'" <firstname.lastname@example.org>, Katri Tuomala <email@example.com>|
|Date:||Wed, 28 Jul 1999 12:41:09|
Indeed, during drying Crystalmount will develop an uneven surface. The
problems associated with this can be easily circumvented by coverslipping
the sections AFTER the crystalmount has dried. Crystalmount actually forms
a film protecting the sections from organic solvents like toluene or
xylene. I don't know about the xylene replacement solutions.
What we do is leave the sections to dry (in a dust-free area !!! seems
obvious but ... ) after covering them with Crystalmount. Once the
crystalmount is completely dry (usually overnight), we just dip the slides
in toluene and apply Eukitt mouting medium and coverslip. The dip in
toluene is not necessary, but allows the Eukitt medium to spread evenly.
There is actually a reason why you should leave the Crystalmount to dry.
The main problem with most aqueous mouting mediums is their refractive
index, which is not as close to glass than that of organic media. This
results in "parasite" contrast upon observation, especially with thick
sections (> 10 microns). The refractive index of Crystalmount increases
upon drying to approach that of glass. Just look at the sections after you
applied Crystalmount and after the Crystalmount has dried. Coverslipping
the slides after drying will improve the quality of the preparation, and
protect the Crystalmount in case of oil immersion observation. The dried
Crystalmount is insoluble in toluene etc, but not in water or ethanol !!
Thus, after immersion observation, if you clean the slide with ethanol ....
the Crystalmount goes !!
We have been using Crystalmount for over a year now for ISH/ICC
doublelabelling using NBT/BCIP and AEC, and the Crystalmount is the only
medium coming close to dehydrating the slides and coverslipping with an
organic solvent based medium, especially with 25-50 micron vibratome or
cryostat sections. And I am not too happy about his, because I still think
that Crystalmount is excessively priced.
A 15:20 27/07/1999 -0500, Nocito, Joseph a écrit :
>please be careful about using Crystalmount only. My experience is that
>after the Crystalmount hardens, it tends to develop an uneven surface. This
>is noticable whe nviewing the slides at 40x or higher. It becomes hard to
>focus up and down. That is why I use a coverslip with the Crystalmount,
>even though the manufacturer says not to (Sometimes I have to go against the
>grain). I have pulled 3-4 year old immuno slides that were stained with AEC
>and to my suprise, the reaction was still there. After everyone told me
>that AEC fades, well, this just blew me away. imagine, 4 year old slides
>stained with AEC and STILL visible. What a concept!!
>Joe Nocito, B.S., HT(ASCP)QIHC
>Christus Santa Rosa Hospitals
>San Antonio, Texas
>> -----Original Message-----
>> From: P. Emry [SMTP:firstname.lastname@example.org]
>> Sent: Tuesday, July 27, 1999 12:35 PM
>> To: Katri Tuomala
>> Cc: Rudman, Dorene M.; 'histonet@Pathology.swmed.edu'
>> Subject: Re: IHC mounting media
>> Hi Katri,
>> Am I reading this right? You just cover the specimen and let it harden?
>> Is there a special way to apply it? Why haven't I heard of this...sounds
>> Trisha, U of WA, Seattle
>> On Tue, 27 Jul 1999, Katri Tuomala wrote:
>> > Hi Doreen,
>> > There are several vendors that carry a permanent aqueous mounting media,
>> > which preserves the staining well. My experience is with Crystalmount
>> > (Biomeda?). It is expensive, but does not require the use of coverslips,
>> > it dries hard. Hope this helps.
>> > Katri
>> > Katri Tuomala
>> > Anatomic Pathology
>> > St.Joseph's Hospital
>> > Hamilton, Ontario, Canada
(o -) O
Paul Klosen, PhD
CNRS UMR 7518 Neurobiologie des Fonctions Rythmiques et Saisonnieres
Universite Louis Pasteur
12, rue de l'Universite
F-67000 Strasbourg, FRANCE
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