[Histonet] (no subject)

[Galina Deyneko]

Hi Subat,
I like the following protocol for mouse hearts ( i received it from James Watson and modified little bit).
70 % Ethanol - optional ( we keep the tissue in 70 % some times coulple days before processing in cold room at 4C)
80% , 95%, 95%- 45 min each run, wacuum off
100%- 45 min
100% with glycerol(5% solution of glycerol)- 45 min
100% with glycerol(5% solution of glycerol)- 45 min
Xylene -30 min,
Xylene -45 min, 
Xylene - 45 min
Paraffin 1, paraffin II, Paraffin III- 45 min each.
Started from 100 % the wacuum is on.
We use snandon processing center.
I am not alone who use a processor that is why we exclude Glycerol, but I am sure that this reagent is goog for processing of the hearts.
Be very patient during the cutting and check the sections in microscope. I soak the specimens in the ice before cutting and you can add fabric softener or several drops Tween 20 into the ice.I like advice to soak a few seconds the trimmed block in hot water before placed on ice. Even you can soak 5 minutes in DI water at RT, but be very careful with the first section, the tissue protrudes after soaking. Also we use "intermediate" water bath with DI water with RT and place the sections in this water bath first and after transfer on glass slide to hot water bath.
Also you can soak with cold water the surface of the block using finger, and I like to breath on the surface of the block before each section, breathing gives some additional moisture to surface and facilitates cutting.
We use two type of paraffin for processing and embedding from Surgipath. 
Also i can give the good reference for paraffin "Shandon precision cut" Thermo Shandon cat # B1002490.
Best regards 
galina deyneko
Novartis Cambridge MA