Day 3
Procedures (T1, S1):
Chill a pipet and gently mix solutions. Using a forceps carefully raise an
ACLAR spear, if there is no sample attached to it, remove it to a rinsing
container. If a sample is still attached, lower it back into its capsule and
gently tap the spear against the bottom and flex it a bit, then check again to
see if the sample has detached. Repeat if necessary, or move on and come
back. Continue until all spears are removed. If desperate, chill a tool and
use it to dislodge the sample. Do a final round of gentle solution mixing,
then close lid, lower glass, and detach it from the lifter.
Day 4
Preparations:
Mix by swirling gently, do not incorporate air bubbles.
5 ml of solution are needed for each holder. Dedicated 20 ml vials with
caps for full strength Lowicryl and for mixtures with methanol are useful.
Keep solutions in dark as much as possible.
For 1:1 Lowicryl/methanol—Make up 20 ml as above, take 8 ml and
combine with 8 ml methanol in mixing bottle, swirl gently to mix.
For 2:1 Lowicryl/methanol—Take remaining 12 ml from the 20 ml above
and combine with 6 ml methanol in mixing bottle.
Dry overnight at 60 °C:
The larger parts of 24 gelatin capsules (Leica 16702745) size 1
Spider covers (to pick up and transfer capsules)
Stem holders for spider covers
Bottom plate (spacer) for cans
4–5 universal cans
Chamber for gelatin capsules
Procedures (T2):
Chill solutions and pipets as in beginning.
Remove uranyl acetate solution (collect for hazardous waste disposal).
3 Â 15 min rinses in methanol, with gentle mixing as before (collect for
disposal).
2 h 1:1 Lowicryl/methanol
Lowicryl HM-20 (ml)
20
15
10
Crosslinker D (g)
2.98
2.24
1.49
Monomer E (g)
17.02
12.76
8.51
Mix gently, add
Initiator C (g)
0.1
0.075
0.05
6 Conjugate Immunofluorescence—SEM Array Tomography …
171
Procedures (T1, S1):
Chill a pipet and gently mix solutions. Using a forceps carefully raise an
ACLAR spear, if there is no sample attached to it, remove it to a rinsing
container. If a sample is still attached, lower it back into its capsule and
gently tap the spear against the bottom and flex it a bit, then check again to
see if the sample has detached. Repeat if necessary, or move on and come
back. Continue until all spears are removed. If desperate, chill a tool and
use it to dislodge the sample. Do a final round of gentle solution mixing,
then close lid, lower glass, and detach it from the lifter.
Day 4
Preparations:
Mix by swirling gently, do not incorporate air bubbles.
5 ml of solution are needed for each holder. Dedicated 20 ml vials with
caps for full strength Lowicryl and for mixtures with methanol are useful.
Keep solutions in dark as much as possible.
For 1:1 Lowicryl/methanol—Make up 20 ml as above, take 8 ml and
combine with 8 ml methanol in mixing bottle, swirl gently to mix.
For 2:1 Lowicryl/methanol—Take remaining 12 ml from the 20 ml above
and combine with 6 ml methanol in mixing bottle.
Dry overnight at 60 °C:
The larger parts of 24 gelatin capsules (Leica 16702745) size 1
Spider covers (to pick up and transfer capsules)
Stem holders for spider covers
Bottom plate (spacer) for cans
4–5 universal cans
Chamber for gelatin capsules
Procedures (T2):
Chill solutions and pipets as in beginning.
Remove uranyl acetate solution (collect for hazardous waste disposal).
3 Â 15 min rinses in methanol, with gentle mixing as before (collect for
disposal).
2 h 1:1 Lowicryl/methanol
Lowicryl HM-20 (ml)
20
15
10
Crosslinker D (g)
2.98
2.24
1.49
Monomer E (g)
17.02
12.76
8.51
Mix gently, add
Initiator C (g)
0.1
0.075
0.05
6 Conjugate Immunofluorescence—SEM Array Tomography …
171