(c) Place one 10 μl droplet of primary antibody, diluted in
PBS-BSA buffer to the concentration of 2 mg/ml, on
paraffin films; then float TEM grids with OVA antigens
side on the top of droplet; followed by incubation in a
wet chamber at RT for 10–15 min.
(d) Wash the grids using 10 μl droplet of PBS buffer
three times.
(e) Incubate the grids in a droplet of 10 μl Au-IgG Ab
diluted in PBS buffer for 5–10 min at RT.
(f) Wash the grids with PBS buffer and then with MilliQ water.
(g) Blot excess liquid and dry TEM grids at RT before
imaging.
II. Labeling SLA archaeosome-based formulations: archaeosomes entrapped with and without OVA. The labeling of
archaeosomes can be done in suspension although this is
suboptimal (see Note 4) and thus labeling on TEM grids is
preferred. For negative control, the steps (b)–(d) below are
not necessary since the sample is labeled with secondary
Au-IgG Ab only.
(a) Place a 6 μl droplet of archaeosome aqueous sample on
a lacey carbon film with ultrathin formvar film
TEM grid.
(b) Place a 2 μl droplet of PBS-BSA buffer on TEM grids
and incubate in a wet chamber for 10 mins at 37
C.
(c) Blot excess liquid; then float TEM grids on a 20 μl
droplet of primary Ab and incubate in a wet chamber
for about 1 h at 37
C.
(d) Wash TEM grids using 10 μl droplet of PBS buffer
three times.
(e) Place a 10 μl droplet of diluted Au-IgG Ab in PBS on
grid for 30 min at RT or 1 h at 4
C chamber (see Note
5).
(f) Wash TEM grids using 10 μl droplet of PBS buffer
three times.
(g) Place 5 μl droplet of PBS on TEM grids which is ready
for the subsequent plunge freezing.
4. Plunge-freeze the archaeosomes on TEM grids (see Note 6).
(a) Precool the cryogen cup in liquid N 2 for about 30 min.
(b) Liquefy the compressed ethane gas into a Falcon tube
surrounded by liquid N 2 .
(c) Pour liquid ethane into cryogen cup and wait until the
bottom of the cup freezes.
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Hui Qian et al.
PBS-BSA buffer to the concentration of 2 mg/ml, on
paraffin films; then float TEM grids with OVA antigens
side on the top of droplet; followed by incubation in a
wet chamber at RT for 10–15 min.
(d) Wash the grids using 10 μl droplet of PBS buffer
three times.
(e) Incubate the grids in a droplet of 10 μl Au-IgG Ab
diluted in PBS buffer for 5–10 min at RT.
(f) Wash the grids with PBS buffer and then with MilliQ water.
(g) Blot excess liquid and dry TEM grids at RT before
imaging.
II. Labeling SLA archaeosome-based formulations: archaeosomes entrapped with and without OVA. The labeling of
archaeosomes can be done in suspension although this is
suboptimal (see Note 4) and thus labeling on TEM grids is
preferred. For negative control, the steps (b)–(d) below are
not necessary since the sample is labeled with secondary
Au-IgG Ab only.
(a) Place a 6 μl droplet of archaeosome aqueous sample on
a lacey carbon film with ultrathin formvar film
TEM grid.
(b) Place a 2 μl droplet of PBS-BSA buffer on TEM grids
and incubate in a wet chamber for 10 mins at 37
C.
(c) Blot excess liquid; then float TEM grids on a 20 μl
droplet of primary Ab and incubate in a wet chamber
for about 1 h at 37
C.
(d) Wash TEM grids using 10 μl droplet of PBS buffer
three times.
(e) Place a 10 μl droplet of diluted Au-IgG Ab in PBS on
grid for 30 min at RT or 1 h at 4
C chamber (see Note
5).
(f) Wash TEM grids using 10 μl droplet of PBS buffer
three times.
(g) Place 5 μl droplet of PBS on TEM grids which is ready
for the subsequent plunge freezing.
4. Plunge-freeze the archaeosomes on TEM grids (see Note 6).
(a) Precool the cryogen cup in liquid N 2 for about 30 min.
(b) Liquefy the compressed ethane gas into a Falcon tube
surrounded by liquid N 2 .
(c) Pour liquid ethane into cryogen cup and wait until the
bottom of the cup freezes.
504
Hui Qian et al.