Re: [Histonet] heart specimens


Your fixation sounds fine. You may want to rethink your time in the
alcohols and Xylene. Animal tissue by itself is dry.

Placing the sample in three 100% alcohols for 45 minutes is really taking
the bound water out of the tissue.  This will account for the dry crumbly
sections. Cut back to two 100% alcohols.

You can also cut back on the Xylene. Two stations would be more than
enough. Too much time in Xylene will make the tissue brittle.

The last thing you may want to consider is the time in each station
including paraffin. Change the timing to no more than 20 - 30  minutes in
each station.

There is an excellent book available through NSH. It has a selection of
processing protocols for animal tissue from several different researchers.
The title is "Aminal Processing Manual". The editors are Gayle Callis and
Diane Sterchi. You will find this book to be very usefull!

Best Regards

Mari Ann Mailhiot BA HT ASCP
Application Specialist
Leica Technical Assistance Center
800 248 0123 x7267
847 236 3063 fax

                      Galina Deyneko                                                                                                                
                      Sent by:                              cc:                                                                                     
                      histonet-bounces@lists.utsouth        Subject:  [Histonet] heart specimens                                                    
                      01/18/2005 04:14 PM                                                                                                           

Galina Deyneko  wrote: Dear colleagues please
I am performing evaluation of the mouse's heart model of cardiac
hypertrophy and infarct, but I ran into some problems with microtome
sectioning of paraffin blocks of the hearts. It is first time in my
practice and I am upset. After harvesting (without perfusion with BNF)
hearts are fixed in 10% BNF (I also tried fixation in Bouin's solution and
4% paraphormaldehyde) for 48 hrs and also the time of fixation was extended
to 1 week, dehydrated overnight in 70% Ethanol in Cold Room, dissected in
short and long axes, and processed in Shandon Processing Center.
Ethanol : 60%- 30min              Xylene1- 45min
              70%-30 min              Xylene2- 45 min
              95%-40 min              Xylene3- 45 min
              95%-40 min               Wax1-   45 min
              100%-40 min              Wax2-   1. hrs
              100%-45 min               Wax3-   1hrs.
              100%-45 min
I tried to increase and decrease time of processing with and without vacuum
and kept in last wax until next morning. Heart tissues appear very dry;
they also crumble during sectioning. Before sectioning  I kept blocks on
ice tray and I also tried to soak them in soap solution. I wetted them with
1% ammonium hydroxide. For waterbath I use distilled water of 43*C. Tissue
sections under objective X2 and X4 look unsatisfactory: muscle fibers are
torn in many places, crumbled, with pieces torn off, but the remain tissue
section mostly without wrinkles. What is significant is that under higher
magnification (X100-X400) the cells and nuclei appear fine and  clear, but
for the photography and measurement my slides are inadequate. Any advice
would be highly appreciated.
Galina Deyneko.

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