Biology Reference
In-Depth Information
2.
Within 24 h, the tissue is being processed. Small pieces of heart tissue are
transferred on a glass slide and further trimmed under the dissecting microscope
in a fume hood. The orientation of the heart muscle bundle is discernible at
10-20
magnification. Using a sharp surgical blade, heart muscle tissue is
trimmed into 1 mm
2 mm pieces with the long axis parallel with the
direction of the myofibril bundles. A transparency film printed with 1 mm
2
gridded pattern can be placed underneath the glass slide as a size guide. A drop
of fixative containing 2% paraformaldehyde and 2.5% glutaraldehyde in
phosphate buffer is added onto the muscle piece to prevent drying during
trimming.
3.
After trimming, tissue pieces left in 2% paraformaldehyde and 2.5%
glutaraldehyde for another 30 min, washed with two changes of 100 mM
phosphate buffer, 2 min each time, quenched in 100 mM glycine in 100 mM
phosphate buffer for 15 min, and washed with phosphate buffer three times,
5 min each time. It is important to completely remove traces of aldehyde
solution before the osmium staining step.
4.
After washing, specimens are postfixed with 1% osmium tetroxide in 100 mM
phosphate buffer for 1 h and washed three times in water (10 min each wash).
5.
After washing, specimens are postfixed with 1% osmium tetroxide in 100 mM
phosphate buffer for 1 h and washed three times in water (10 min each wash).
6.
Specimens are then
en bloc
stained with 3% UA in deionized water for 1 h at
room temperature and washed three times in deionized water (10 min each
wash).
7.
This is followed by dehydration in a graded ethanol series including incubation
in 30, 50, 70, 90% ethanol (10 min each), and two successive 10 min
incubations in 100% ethanol. Care should be taken that incubation of the
specimen in ethanol is not prolonged to minimize the chances of lipid
extraction.
8.
Tissue pieces are then infiltrated with 1:1 ethanol/spurs resin, 2:1 ethanol/spurs
resin for 1 h each, and pure spurs resin overnight.
9.
The following morning, specimens are transferred to a new tube containing pure
spurs resin for 1 h and then transferred with a wooden toothpick into a flat
embedding mold with either the long side of the specimen placed perpendicular
or parallel to the tip of the mold.
10.
Polymerization of the resin is achieved at 60
C for 24-48 h. The embedded
blocks are trimmed as a trapezoid with the parallel sides at
1mm
0.5-1 mm in size.
Survey sections of 150-250 nm thickness are collected onto glass slides, and
stained with toluidine blue to confirm the orientation of the myofibrils:
longitudinal or transversal.
11.
Judging from the orientation of the myofibrils, the angle of the resin block can
be adjusted in order to obtain the largest possible area of sections with a
longitudinal cut of the muscle fibers. Silver-colored ultrathin sections at
70 nm thickness are collected onto 200 mesh Cu grids and directly viewed in
the electron microscope without further uranyl acetate or lead poststaining.
