Re: [Histonet] Excessively wrinkled sections

From:"Fred Underwood"

I am dealing with human hearts, but I'll throw in my 2 cents.  You could
safely raise your water bath temp. to a range of 43 to 45 degrees C. 
Much higher than that and the paraffin begins to melt on the water.  An
aid that I use constantly is to have some kind of hot plate ( one used
in making stains or solutions will work fine) and adjust the temperature
to 55 degrees C, or so.  Quickly touch the slide ( with section mounted)
to the hotplate, this will help pull out wrinkles.  It will take you a
few times of trial and error to get the correct temp. and technique, but
it works great.  Also, I limit the amount of contact with water while
the blocks are cooling, that is if you face your blocks prior to


>>> "Barlow, Gillian"  09/25/03 02:00PM >>>
Dear Hisonetters

First, many thanks to those of you who replied to help me with my
fixations - I now have nice, well-fixed tissues.  However, one problem
remains - the sections are very wrinkled and do not flatten out well on
waterbath, even when left for long periods of time.  I store my blocks
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
dont want to damage my antigen.

Many thanks

PS  For anyone out there with questions on cardiac fixation and
this is the protocol I have now found to work well and wanted to share:

Hearts were isolated from anaesthetized animals, cut in half and fixed
overnight in 10% buffered formalin at room temperature.  The following
hearts were transferred to 70% ethanol and stored at 4 degrees prior

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

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