Immunogold labeling for electron microscopy
This protocol is extracted from research article:
Horizontal genome transfer by cell-to-cell travel of whole organelles
Sci Adv, Jan 1, 2021; DOI: 10.1126/sciadv.abd8215

For immunogold labeling, cryo-fixed and freeze-substituted samples embedded into LR White were cut into 100-nm-thin sections. Nonspecific antibody binding was reduced by incubation of samples in blocking buffer [phosphate-buffered saline–Tween 20 (PBST) containing 2% bovine serum albumin and 0.1% fish gelatin; Sigma-Aldrich] for 1 hour at room temperature. Antigens were detected by incubation in blocking buffer containing anti-dsRed (1:100 dilution, mouse; ChromoTek GmbH, Planegg-Martinsried, Germany), anti-ClpP (1:200, rabbit; provided by A. Clarke, University of Gothenburg, Sweden), anti-RbcL (1:200, rabbit; Agrisera, Vännäs, Sweden), or anti-GLN2 (1:200, rabbit; Agrisera) antibodies for 1 hour at room temperature. Excess antibodies were removed by six rinses with PBST buffer for 3 min each. Following hybridization to sections, bound primary antibodies were detected by incubation for 1 hour in blocking buffer containing 10 nm (for mouse; Cell Signaling Technology, Danvers, MA) or 25 nm colloidal gold-labeled secondary antibodies (goat anti-rabbit antibody, 1:20; AURION, Wageningen, The Netherlands). After six rinses with PBST and three rinses with double-distilled water for 3 min each, samples were contrasted with aqueous 2% uranyl acetate for 12 min and Reynolds’ lead citrate for 7 min.

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