Pellet is washed twice with pyrogen-free water and resuspended with PBS buffer to desired volume.
(b) Empty archaeosomes are formed using the same processes
above except that lipid film is hydrated in Milli-Q water
without protein antigen.
3.2 Cryo-TEM
Specimen Preparation
1. Select proper TEM grids to meet different imaging and analysis
requirements (see Note 1). Continuous ultrathin carbon film
supported TEM grids are used for negative staining or immunogold labeling antigens at room temperature in dried state.
Lacey carbon film TEM grids are used for cryo-EM specimens.
The lacey carbon film with an ultrathin formvar film Nickle
grids are used for cryo-EM specimens with immunogold
labeling.
2. Glow discharge TEM grids (see Note 2). Place the carbon side
of TEM grids facing up in the discharge chamber. Set the
pressure in glow chamber to 40 mbar at air atmosphere, discharging current of 15–20 μA and discharging time of 10–20 s.
3. Immunogold labeling archaeosomes based formulations (see
Note 3).
I. Labeling ovalbumin as positive control to check the effectiveness of immunogold labeling (Fig. 4).
(a) Dilute aliquot OVA with 1Â PBS to the concentration
of 1 mg/ml. Dilute Au-IgG Ab 40Â (1:40) in PBS
buffer.
(b) Place 4 μl of OVA aqueous solution on continuous
carbon TEM grids and blot excess liquid after 1 min.
Fig. 4 Bright field TEM image of OVA antigens immunolabeled with primary Ab
and 1.4 nm nanogold conjugated secondary Ab. Aggregates marked as white
arrows
Cryo-TEM for Vaccine Delivery Carrier Evaluation
503
(b) Empty archaeosomes are formed using the same processes
above except that lipid film is hydrated in Milli-Q water
without protein antigen.
3.2 Cryo-TEM
Specimen Preparation
1. Select proper TEM grids to meet different imaging and analysis
requirements (see Note 1). Continuous ultrathin carbon film
supported TEM grids are used for negative staining or immunogold labeling antigens at room temperature in dried state.
Lacey carbon film TEM grids are used for cryo-EM specimens.
The lacey carbon film with an ultrathin formvar film Nickle
grids are used for cryo-EM specimens with immunogold
labeling.
2. Glow discharge TEM grids (see Note 2). Place the carbon side
of TEM grids facing up in the discharge chamber. Set the
pressure in glow chamber to 40 mbar at air atmosphere, discharging current of 15–20 μA and discharging time of 10–20 s.
3. Immunogold labeling archaeosomes based formulations (see
Note 3).
I. Labeling ovalbumin as positive control to check the effectiveness of immunogold labeling (Fig. 4).
(a) Dilute aliquot OVA with 1Â PBS to the concentration
of 1 mg/ml. Dilute Au-IgG Ab 40Â (1:40) in PBS
buffer.
(b) Place 4 μl of OVA aqueous solution on continuous
carbon TEM grids and blot excess liquid after 1 min.
Fig. 4 Bright field TEM image of OVA antigens immunolabeled with primary Ab
and 1.4 nm nanogold conjugated secondary Ab. Aggregates marked as white
arrows
Cryo-TEM for Vaccine Delivery Carrier Evaluation
503