[Histonet] Decloaker
Orr, Rebecca
ROrr <@t> enh.org
Fri Oct 28 13:53:04 CDT 2005
Wen,
I have used the Biocare Decloaker for several years.
In my experience, I would agree 20 minutes is really too long, at such a
high temperature.
You will most likely get staining, but you will most definitely loose
tissue as well as morphology.
I know of labs like Jan Shivers' at U of MN that use the decloaker to
test prions and that has an extended time at the 120'-125'C. So there
are applications where this may be beneficial to you...
But clinically speaking, if you have to work that hard, something's
wrong somewhere else! I understand research and non hospital settings
have different needs and unique antibodies...but if I would have to use,
in my opinion, such extreme parameters, I would try a different ph
solution, or look at the enzymes to digest or even a stronger dilution.
Then there's always fixation issues...
>From what I get from your email is that you asked for directions to
extend the time at the high temperature. I'm sure Biocare Tech support
instructed you how to make the change with the recommendation that you
shouldn't really need that, for HEIR. You've gotten a bunch of our
opinions, as to why you shouldn't need that high a temp, but regardless,
it's your lab and your test and your responsibility...so have at it! And
let us know your results!
As to how to set the program, I see others have shared.
Good luck, I'd be very interested to hear of your results.
Becky Orr CLA,HT(ASCP)
IHC Lead
Evanston Northwestern Healthcare
847-570-2771
> Hello,
>
> Does anybody use Decloaking Chamber from Biocare Medical? How should I
set the
> SP1 temperature and time if I need 20 minutes treatment? The factory
setting
> is 125 degree 30 seconds and the tech support said 20 minutes is too
long. She
> suggested me to keep 30 seconds. I still want the suggestion from
here. How do
> you set your program?
>
> Thanks!
> Wen
>
>
> ---------------------------------
> Yahoo! FareChase - Search multiple travel sites in one click.
>
> ------------------------------
>
> Message: 2
> Date: Thu, 27 Oct 2005 11:30:02 -0600
> From: Gayle Callis <gcallis <@t> montana.edu>
> Subject: [Histonet] Help!! We have a problem with a Mounting Media
> To: Histonet <@t> lists.utsouthwestern.edu
> Message-ID:
> <6.0.0.22.1.20051027111608.01b0de68 <@t> gemini.msu.montana.edu>
> Content-Type: text/plain; charset="us-ascii"; format=flowed
>
> Is anyone experiencing this problem with Protocol Mounting Medium T,
> toluene solvent based from Fisher.
>
> They take 50 mls and put it into a bottle with a secure, air tight,
solvent
> resistant lid in order to not dip into stock bottle all the time.
> If they use the mounting media absolutely fresh after aliquoting, the
> mounting media drys nicely, everything is fine. They are
coverslipping
> cytospin stained blood cells, Diff Quik stained, and dry without any
> immersion oil used before coverslipping.
>
> If they let the Protocol media aliquot, even though in an air tight,
new
> bottle, sit around, they notice a layer or some type of separation
within
> top layer of media. When they use this now separated aliquot,
stirred,
> and then try to dry slide, the coverslip still slips and slides
around -
> failure of drying!! Very messy!
>
> Something is happening, either evaporation of toluene to change ratio
of
> solvent to acrylic resin, maybe something else i.e faulty
> manufacturing/preparation of this media? This is a total mystery to
me as
> I have done this type of aliquoting with other mounting medias for
years
> and NEVER had a problem except to add solvent to restore media - thick
to
> thinner. Nor I have never experienced layering/separation phenomena
with
> my other mounting media (Richard Allan brand or Permount). After
> coverslipping, the latter mounting media set up and dry nicely with no
> slip/sliding away!!!
>
>
>
> Gayle Callis
> Research Histopathology Supervisor
> Veterinary Molecular Biology
> Montana State University - Bozeman
> PO Box 173610
> Bozeman MT 59717-3610
> 406 994-6367
> 406 994-4303 (FAX)
>
>
>
>
>
> ------------------------------
>
> Message: 3
> Date: Thu, 27 Oct 2005 10:31:19 -0700 (PDT)
> From: Kim Merriam <kmerriam2003 <@t> yahoo.com>
> Subject: [Histonet] mouse heart matrix
> To: Histonet <histonet <@t> lists.utsouthwestern.edu>
> Message-ID: <20051027173119.75498.qmail <@t> web50310.mail.yahoo.com>
> Content-Type: text/plain; charset=iso-8859-1
>
> Hello all,
>
> I was wondering if anyone knew of a vendor that sells a tissue matrix
that
> will allow me to take thin cross-sections of mouse heart (something
similar to
> the brain matrices sold by Ted Pella.
>
> Regards,
> Kim
>
>
> Kim Merriam
> Novartis
> Cambridge, MA
>
> ---------------------------------
> Yahoo! FareChase - Search multiple travel sites in one click.
>
> ------------------------------
>
> Message: 4
> Date: Thu, 27 Oct 2005 12:34:37 -0500
> From: "Jackie M O'Connor" <Jackie.O'Connor <@t> abbott.com>
> Subject: Re: [Histonet] Decloaking Chamber (high pressure cooker)
> To: wen eng <weneng2004 <@t> yahoo.com>
> Cc: histonet <histonet <@t> lists.utsouthwestern.edu>
> Message-ID: <OF4EC23582.183E56C2-ON862570A7.006039B1 <@t> abbott.com>
> Content-Type: text/plain; charset="us-ascii"
>
> I'm using the factory settings for virtually all antibodies - so far,
I
> haven't found the need to change the settings for anything. I did
uncover
> a problem, however, with the computer chip in my old Decloaker - the
> temperature and pressure were not reaching the set point - but I
didn't
> notice until I sat down to actually watch the machine. The electronic
> temperature display was bouncing all over the place. I had to get a
new
> machine.
> Jackie O'
>
>
>
>
>
> wen eng <weneng2004 <@t> yahoo.com>
> Sent by: histonet-bounces <@t> lists.utsouthwestern.edu
> 10/27/2005 12:27 PM
>
>
> To: histonet <histonet <@t> lists.utsouthwestern.edu>
> cc: (bcc: Jackie M O'Connor/LAKE/GPRD/ABBOTT)
> Subject: [Histonet] Decloaking Chamber (high pressure
cooker)
>
>
> Hello,
>
> Does anybody use Decloaking Chamber from Biocare Medical? How should I
set
> the SP1 temperature and time if I need 20 minutes treatment? The
factory
> setting is 125 degree 30 seconds and the tech support said 20 minutes
is
> too long. She suggested me to keep 30 seconds. I still want the
suggestion
> from here. How do you set your program?
>
> Thanks!
> Wen
>
>
> ---------------------------------
> Yahoo! FareChase - Search multiple travel sites in one click.
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
>
>
> ------------------------------
>
> Message: 5
> Date: Thu, 27 Oct 2005 12:43:23 -0500
> From: "Sebree Linda A." <la.sebree <@t> hosp.wisc.edu>
> Subject: RE: [Histonet] Decloaking Chamber (high pressure cooker)
> To: "wen eng" <weneng2004 <@t> yahoo.com>, "histonet"
> <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
> <D6B654003615874B873E15BA680E2D2212FD12A7 <@t> uwhis-xchng1.hosp.wisc.edu>
> Content-Type: text/plain; charset="us-ascii"
>
> We translated 20 minutes HIER in a water bath to around 2-3 minutes in
> the Decloaking Chamber. Hope this helps.
>
> Linda A. Sebree
> University of Wisconsin Hospital & Clinics
> IHC/ISH Clinical & Research Laboratory
> DM223-VA
> 600 Highland Ave.
> Madison, WI 53792
> (608)265-6596
> FAX: (608)262-7174
>
>
>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of wen
eng
> Sent: Thursday, October 27, 2005 12:28 PM
> To: histonet
> Subject: [Histonet] Decloaking Chamber (high pressure cooker)
>
>
> Hello,
>
> Does anybody use Decloaking Chamber from Biocare Medical? How should I
> set the SP1 temperature and time if I need 20 minutes treatment? The
> factory setting is 125 degree 30 seconds and the tech support said 20
> minutes is too long. She suggested me to keep 30 seconds. I still want
> the suggestion from here. How do you set your program?
>
> Thanks!
> Wen
>
>
> ---------------------------------
> Yahoo! FareChase - Search multiple travel sites in one click.
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
>
> ------------------------------
>
> Message: 6
> Date: Thu, 27 Oct 2005 13:01:35 -0500
> From: "Jan Shivers" <shive003 <@t> umn.edu>
> Subject: Re: [Histonet] Decloaking Chamber (high pressure cooker)
> To: "wen eng" <weneng2004 <@t> yahoo.com>
> Cc: histonet <histonet <@t> lists.utsouthwestern.edu>
> Message-ID: <005901c5db20$78b82e60$41065486 <@t> auxs.umn.edu>
> Content-Type: text/plain; charset="iso-8859-1"
>
> I use a Decloaking Chamber for Chronic Wasting Disease slides, and
yes, 20
> minutes is the correct setting for that particular test, with a 25
minute
> cooldown. Most other IHC tests don't require a lengthy setting on the
> Decloakers.
>
> I'd recommend contacting Biocare to do this for you. They came and
> installed the settings on my chambers.
>
> Jan Shivers
> U of MN Vet Diag Lab
>
> ----- Original Message -----
> From: "wen eng" <weneng2004 <@t> yahoo.com>
> To: "histonet" <histonet <@t> lists.utsouthwestern.edu>
> Sent: Thursday, October 27, 2005 12:27 PM
> Subject: [Histonet] Decloaking Chamber (high pressure cooker)
>
>
>> Hello,
>>
>> Does anybody use Decloaking Chamber from Biocare Medical? How should
I set
> the SP1 temperature and time if I need 20 minutes treatment? The
factory
> setting is 125 degree 30 seconds and the tech support said 20 minutes
is too
> long. She suggested me to keep 30 seconds. I still want the suggestion
from
> here. How do you set your program?
>>
>> Thanks!
>> Wen
>>
>>
>> ---------------------------------
>> Yahoo! FareChase - Search multiple travel sites in one click.
>> _______________________________________________
>> Histonet mailing list
>> Histonet <@t> lists.utsouthwestern.edu
>> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>>
>
>
>
>
> ------------------------------
>
> Message: 7
> Date: Thu, 27 Oct 2005 14:24:14 -0500
> From: "Johnson, Teri" <TJJ <@t> Stowers-Institute.org>
> Subject: [Histonet] Re: Red HRP chromagen/substrate
> To: <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
>
<C28BAF593DC3314E9C0F3A50191C2E78016CB830 <@t> EXCHKC03.stowers-institute.org
>
>
> Content-Type: text/plain; charset="us-ascii"
>
> Andrea,
>
> It depends on what you consider "best". As Gayle pointed out, for
some
> it's endpoint color. For others, it's permanence. AEC needs to be
> mounted in aqueous mountant because it's not stable in organic
solvents
> (even the polyvinyl alcohol in some of the aqueous mountants can cause
> fading!). The way to get around this is to either use a permanent
> chromogen (Vector Nova Red), or you can use Crystal mount to cover the
> AEC stained tissue sample and allow to dry. We always coverslip it
with
> permanent mountant for better visualization under the microscope.
>
> Teri Johnson
> Managing Director Histology Facility
> Stowers Institute for Medical Research
> 1000 E. 50th St.
> Kansas City, MO 64133
>
>
>
> ------------------------------
>
> Message: 8
> Date: Thu, 27 Oct 2005 15:36:27 -0400
> From: "John A. Kiernan" <jkiernan <@t> uwo.ca>
> Subject: Re: [Histonet] Cholesterol staining suggestions
> To: Gayle Callis <gcallis <@t> montana.edu>
> Cc: Histonet <@t> lists.utsouthwestern.edu
> Message-ID: <43612C3B.BD853396 <@t> uwo.ca>
> Content-Type: text/plain; charset=us-ascii
>
> The only specific method for cholesterol that
> doesn't involve a nasty acid is the cholesterol
> oxidase-DAB method (Emeis et al 1977 Histochem. J.
> 9:197-204). It can also detect cholesterol esters
> if the sections are first treated with cholesterol
> esterase. It's rather more than a "stain" but should
> be OK for research. It can also be used at the EM
> level (eg Pelletier & Vitale 1994 J Histochem Cytochem
> 42:1539-1554.)
>
> Regarding the traditional stains for cholesterol:
> According to Adams, Pearse and Bayliss High, the
> perchloric acid-naphthoquinone method is the most
> specific and sensitive one for cholesterol and its
> esters. I've tried it once or twice; it works but
> it's rather rough on the sections. The blue colour
> fades after a few hours. An older, simpler method
> is that of Schultz (here summarized from Bayliss High's
> 1984 "Lipid Histochemistry" p.40-41). I've not tried
> it myself.
>
> Frozen sections, after formal-calcium fixation,
> air-dried onto slides.
> 1. Oxidize sections in 2.5% iron alum in 0.2M acetate
> buffer, pH 3 for 4 hours at 37C.
> 2. Wash, 3 X 20 min in the acetate buffer.
> 3. Rinse in distilled water.
> 4. Treat with 5% formalin in tap water
> (ie 2% formaldehyde), 10 min.
> 5. Drain and allow to dry.
> 6. Apply a drop or two of Schulz reagent (see below),
> and then a coverslip. Examine with microscope.
> Result. Cholesterol blue-green, fading quickly.
> Schulz reagent: Add 1ml glacial acetic acid dropwise to
> 1 ml conc. sulphuric acid, with agitation. It becomes hot
> and syrupy; let it cool.
>
> Cholesterol and its esters are birefringent with crossed
> polars. This property was discovered by B. Doinikow (1913)
> Deutsche Z. Nervenheilk. 46:20-42) and it has been exploited
> to trace degenerating myelinated axons in frozen sections of
> human brain (Miklossy & Van der Loos 1987 Brain Res.
> 426:377-380).
> I tried to reproduce their results in the 1990s but the
> sections contained great quantities of birefringent dirt
> (completely invisible with ordinary illumination). The dirt
> didn't consist of tiny needle-like crystals like the ones
> described by Doinikow and shown in the colour photos in
> Miklossy's paper. My material had been stored for a long
> time
> in formalin before cutting, so perhaps lipids everywhere
> had changed into some sort of birefringent junk.
------------------------------
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