[Histonet] Excessively wrinkled sections

[Gudrun Lang]

----- Original Message -----
From: "Gudrun Lang" <g.lang <@t> bigfoot.de>
To: "Barlow, Gillian" <Gillian.Barlow <@t> cshs.org>
Sent: Monday, September 29, 2003 11:20 AM
Subject: Re: [Histonet] Excessively wrinkled sections


> Hi Gillian,
> I think the discussion about waterbath temperature is interesting, but the
> real  problem is, why are the sections very wrinkled.
> In my opinion heart tissue is not so difficult to cut. We use sliding
> microtoms, 3 um, Aqua dest. waterbath at 40 degrees. And if there are
> wrinkles, where paraffin adhere to paraffin, my experience showed me, that
> in a warm waterbath it never can be seperated and flattend ( only in cold
> water with a brush).
> I don't know if you are a beginner on cutting or have long experience.
> So my advice is to check your cutting-angle, the sharpness of the knife,
the
> roughness of the tissue and don't try too long on one block (temperature).
> good luck
> Gudrun Lang
>
> ----- Original Message -----
> From: "Barlow, Gillian" <Gillian.Barlow <@t> cshs.org>
> To: "'Histonet'" <histonet <@t> lists.utsouthwestern.edu>
> Sent: Thursday, September 25, 2003 8:00 PM
> Subject: [Histonet] Excessively wrinkled sections
>
>
> > Dear Hisonetters
> >
> > First, many thanks to those of you who replied to help me with my
cardiac
> > fixations - I now have nice, well-fixed tissues.  However, one problem
> till
> > remains - the sections are very wrinkled and do not flatten out well on
> the
> > waterbath, even when left for long periods of time.  I store my blocks
at
> > room temperature, but put them on ice for at least two hours before
> > sectioning.  The waterbath is at 40 degrees.  A colleague suggested
> > increasing the waterbath temperature, but the sections are for IHC and I
> > dont want to damage my antigen.
> >
> > Many thanks
> > Gillian
> >
> > PS  For anyone out there with questions on cardiac fixation and
embedding,
> > this is the protocol I have now found to work well and wanted to share:
> >
> > Fixation:
> > Hearts were isolated from anaesthetized animals, cut in half and fixed
> > overnight in 10% buffered formalin at room temperature.  The following
> day,
> > hearts were transferred to 70% ethanol and stored at 4 degrees prior to
> > embedding.
> >
> > Embedding:
> > 80% EtOH, 45 mins room temperature
> > 95% EtOH, 45 mins room temperature, two changes
> > 100% EtOH, 45 mins room temperature, two changes
> > Xylene, 45 mins room temperature, two changes
> > Paraffin, 45 mins, 60 degrees under vacuum, two changes
> > Embed immediately
> >
> >
> >
> > Gillian Barlow, PhD
> > Postdoctoral Fellow
> > Laboratory of Julie Korenberg, PhD, MD
> > Cedars-Sinai Medical Center
> > Davis Bldg, Lab 2007
> > 110 George Burns Rd
> > Los Angeles, CA 90048
> >
> > Phone: (310) 423 7650
> > Fax: (310) 423 0302
> >
>