Day 1
Preparations:
0.1 M sodium acetate (NaA): 1.36 g sodium acetate trihydrate/100 ml
distilled water, store at 4 °C
0.1% CaCl 2 : Make a 10% stock solution in water, store at 4 °C; dilute
1:100 with NaA as needed
Glycerol Dilutions (keep at 4 °C):
10% in NaA
20% in NaA
30% in NaA
Substitution solution: 1.5–4% (varies, depending on lot) uranyl acetate in
anhydrous methanol, make day before needed, store tightly capped at 4 °
C, filter (0.2 µm PES 13 mm syringe filter); standard run uses 15 ml
(5 ml/can)
Pre-clean Flo-through capsules (put capsules in a can with absolute ethanol or
methanol, swirl, then remove one by one with forceps and force solvent
through capsule with pipet before setting on Kimwipe to dry). Do the same for
capsule holders, ACLAR spears (2 cm  2 mm), and cans (for sections:
baskets and ACLAR strips—2 cm  6–8 mm), dry overnight in 60 °C oven.
Procedures:
Select sections and put in labeled shell vials with 0.1 M NaA, keep all
chilled, we set the vials in aluminum vial holders on shipping cold packs in
a Styrofoam box on a gently rotating shaker.
Pretreatment:
3 Â 10 min changes of NaA
1 h in 0.1% CaCl 2 /NaA
3 Â 5 min rinses in NaA
Cryoprotection, keep chilled throughout:
30 min in 10% glycerol/NaA
30 min in 20% glycerol/NaA
30 min in 30% glycerol/NaA
Overnight in 30% glycerol/NaA
Day 2
Procedures:
Prepare AFS unit by filling with liquid nitrogen, insert black-topped tube,
turn on and set the following program (can be modified):
T1 = −90 °C for 32 h
S1 = +4 °C/h
T2 = −45 °C for 50 h
S2 = + 5 °C/h
T3 = 0 °C for 40 h
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Preparations:
0.1 M sodium acetate (NaA): 1.36 g sodium acetate trihydrate/100 ml
distilled water, store at 4 °C
0.1% CaCl 2 : Make a 10% stock solution in water, store at 4 °C; dilute
1:100 with NaA as needed
Glycerol Dilutions (keep at 4 °C):
10% in NaA
20% in NaA
30% in NaA
Substitution solution: 1.5–4% (varies, depending on lot) uranyl acetate in
anhydrous methanol, make day before needed, store tightly capped at 4 °
C, filter (0.2 µm PES 13 mm syringe filter); standard run uses 15 ml
(5 ml/can)
Pre-clean Flo-through capsules (put capsules in a can with absolute ethanol or
methanol, swirl, then remove one by one with forceps and force solvent
through capsule with pipet before setting on Kimwipe to dry). Do the same for
capsule holders, ACLAR spears (2 cm  2 mm), and cans (for sections:
baskets and ACLAR strips—2 cm  6–8 mm), dry overnight in 60 °C oven.
Procedures:
Select sections and put in labeled shell vials with 0.1 M NaA, keep all
chilled, we set the vials in aluminum vial holders on shipping cold packs in
a Styrofoam box on a gently rotating shaker.
Pretreatment:
3 Â 10 min changes of NaA
1 h in 0.1% CaCl 2 /NaA
3 Â 5 min rinses in NaA
Cryoprotection, keep chilled throughout:
30 min in 10% glycerol/NaA
30 min in 20% glycerol/NaA
30 min in 30% glycerol/NaA
Overnight in 30% glycerol/NaA
Day 2
Procedures:
Prepare AFS unit by filling with liquid nitrogen, insert black-topped tube,
turn on and set the following program (can be modified):
T1 = −90 °C for 32 h
S1 = +4 °C/h
T2 = −45 °C for 50 h
S2 = + 5 °C/h
T3 = 0 °C for 40 h
6 Conjugate Immunofluorescence—SEM Array Tomography …
169