Biology Reference
In-Depth Information
6.
Uranyl acetate: 2.0% (w/v) in ethanol. Add 1 g of uranyl acetate to 50 mL of freshly
made 50% ethanol. Cover with foil and stir overnight. Store solution at 4
C.
7.
Lead citrate: add chemicals in distilled water in the following order: lead nitrate
1.33 g, sodium citrate dihydrate 1.76 g, 1 N NaOH 5 mL, freshly made boiled
distilled water 30 mL, stir for 10 min to dissolve and add additional 15 mL of
distilled water. Solution becomes cloudy when sodium citrate is added, and clear
when NaOH is added. Store solution at 4
C.
5.2.2
Methods
1.
Double fixation
Pellet 1-2 mL cells at 2000
L
TAP medium, add equal volume of primary fixative to the tube (2.5% glutaraldehyde
final), and incubate 2 h at room temperature with occasional mixing.
Pellet the cells as above, resuspend the pellet in minimal volume of 2-3% low gel
temperature agarose (premelted and cooled to 37-40
C), then dice the agarose/cells
block into 1 mm cubes after solidifying.
Rinse the cubes in phosphate buffer three times each for 15 min to completely
remove the glutaraldehyde, postfix with 1% osmium tetroxide in phosphate buffer
for 2 h at room temperature or 4
C overnight. Cells will turn black, agarose will
not. Then wash blocks three times for 15 min each with phosphate buffer.
g
for 5 min in an Eppendorf tube, resuspend in 500
m
2.
Dehydration
Dehydrate to graded ethanol series, 30%, 50%, 70%, 80%, 90%, and 95% ethanol
(v/v) for 15 min each and then through 100% ethanol twice, 20 min each, 100% pro-
pylene oxide twice, each 15 min. Remove propylene oxide from the tube, leaving a
little solution in the tube so the cells will not dry out.
3.
Infiltration
Replace propylene oxide with the 1:1 mixture of EMBed-812 resin:propylene oxide
and infiltrate 3 h to overnight.
Change to 2:1 mixture of resin:propylene oxide, 8 h to overnight recommended.
Replace with 100% resin, let infiltrate 2-3 days before embedding, replace with
fresh resin once a day.
All the infiltration steps are performed at room temperature and a specimen ro-
tator is set at the lowest speed.
4.
Embedding
Embed cells/agarose cubes in flat embedding molds or embedding capsules, and
bake in 60
C oven for 24 h.
5.
Sectioning
