Immunogold labeling was performed as previously described (B�r�ny et�al. 2010 (link)). Nickel grids carrying ultrathin Lowicryl sections were sequentially floated in PBS, 5% BSA in PBS (w/v), and incubated with rabbit polyclonal anti-ATG8 (Abcam, Cambridge, UK, Cat. ab77003), diluted 1:100 (v/v) in 1% BSA in PBS for 1 h. After several washes in PBS, grids were incubated with anti-rabbit IgG conjugated to 10-nm gold particles (Biocell, Cardiff, UK) diluted 1:25 in PBS, for 1 h at room temperature, washed in PBS and water and air-dried. Ultrathin sections were counterstained with uranyl acetate and lead citrate and observed under a JEOL 1400 TEM at 80 kV. Controls were performed excluding the primary antibody.
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Immunogold Labeling of ATG8 Protein
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Corresponding Organization : Centro de Investigaciones Biológicas Margarita Salas
Other organizations : Swedish University of Agricultural Sciences
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Variable analysis
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- Incubation with rabbit polyclonal anti-ATG8 primary antibody, diluted 1:100 (v/v) in 1% BSA in PBS for 1 h
- Immunogold labeling of ATG8 protein
- Nickel grids carrying ultrathin Lowicryl sections
- Sequential floating in PBS, 5% BSA in PBS (w/v)
- Incubation with anti-rabbit IgG conjugated to 10-nm gold particles, diluted 1:25 in PBS, for 1 h at room temperature
- Counterstaining of ultrathin sections with uranyl acetate and lead citrate
- Observation under a JEOL 1400 TEM at 80 kV
- Positive control: Incubation with rabbit polyclonal anti-ATG8 primary antibody
- Negative control: Excluding the primary antibody
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