[Histonet] heart specimens

[Galina Deyneko]

Galina Deyneko <galinadeyneko <@t> yahoo.com> wrote: Dear colleagues please help!
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.
Sincerely,
Galina Deyneko.


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