Immunogold Labeling and Electron Microscopy

High-pressure freezing (HPF), freeze substitution, ultra-thin sectioning, and imaging were performed as previously described (64 (link)). Immunogold labeling for EM was performed with anti-GFP at optimized concentrations, then with goat anti-rabbit 10 nm gold-coupled secondary antibodies at 1:40 dilution. TEM examination was performed with a Hitachi H-7650 transmission electron microscope with a charge-coupled device (CCD) camera operating at 80 kV (Hitachi High-Technologies). ET, 3D reconstruction, and modeling were performed as described previously (63 (link), 64 (link)).

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Li B., Niu F., Zeng Y., Tse M.K., Deng C., Hong L., Gao S., Lo S.W., Cao W., Huang S., Dagdas Y, & Jiang L. (2023). Ufmylation reconciles salt stress-induced unfolded protein responses via ER-phagy in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, 120(5), e2208351120.

Publication 2023

H-7650

Anti antibodies Device Dilution Freeze substitution Goat Gold Pressure Rabbit Reconstruction Transmission electron microscope

Corresponding Organization :

Other organizations : Chinese University of Hong Kong, Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Chinese University of Hong Kong, Shenzhen

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Variable analysis

independent variables

High-pressure freezing (HPF)
Freeze substitution
Ultra-thin sectioning
Immunogold labeling for EM with anti-GFP at optimized concentrations
Goat anti-rabbit 10 nm gold-coupled secondary antibodies at 1:40 dilution

dependent variables

Imaging
TEM examination with a Hitachi H-7650 transmission electron microscope with a charge-coupled device (CCD) camera operating at 80 kV

control variables

None explicitly mentioned

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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