Tecnai 120kv transmission electron microscope

The tissue samples were collected and fixed in 3% glutaraldehyde.
Processing was done with a Leica EM TP processor (Leica Microsystems Inc., North
Deerfield, IL). Samples were rinsed with buffer, post-fixed in 2% osmium
tetroxide in phosphate buffer, rinsed in distilled water and dehydrated in an
ethanol series transitioning into propylene oxide. The samples were then
infiltrated with Poly/Bed 812 epoxide resin (Polysciences). After
polymerization, selected blocks were trimmed and semithin sections
(approximately 0.5 μm thick) were cut, mounted on glass slides, stained
with 1% toluidine blue in 1% sodium borate, and examined with a light microscope
to ascertain areas of interest. After trimming block faces down to areas of
interest, ultrathin sections (70–90 nm thick) were subsequently cut,
placed on 100 mesh formvar copper grids and stained with uranyl acetate and lead
citrate. Digital images were captured with an Olympus Mega View III side mount
camera attached to an FEI Tecnai 120KV transmission electron microscope.

Selected samples of frozen liver (n=8; 4 high-dose and 4 control animals from both high-fat diet and Control diet groups) were post fixed in 1% osmium tetroxide, then dehydrated through a series of graded alcohols, followed by 100% propylene oxide. Fixed tissues were embedded in Polybed 812 resin and placed in a 70°C oven for 72 hours. The resin blocks were trimmed, thick-sectioned (a 700 to 800nm section stained with 1% Toluidine blue) and examined by light microscopy. One block each from Zone 1 (periportal) and Zone 3 (centrilobular) of the liver was chosen for each animal. The chosen blocks were thin-sectioned (a section 70 to 90 nm or “gold”), placed on formvar-coated copper grids and stained with uranyl acetate and lead citrate. The grids were examined on a FEI Tecnai 120KV transmission electron microscope. At least six digital photomicrographs were taken of each grid/area of the liver (periportal or centrilobular)/animal then evaluated and compared by dose group and hepatic zone.