[Histonet] Excessively wrinkled sections

From:"Barlow, Gillian"

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

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: 

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

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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