Anti atg8

Semithin sections on APTES-coated slides were blocked by 5% (w/v) bovine serum albumin (BSA) (Sigma) in PBS for 10 min, washed with PBS and incubated for 1 h with primary rabbit polyclonal anti-ATG5 (B�r�ny et�al. 2018 (link)) or anti-ATG8 (Abcam, Cambridge, UK, Cat. ab77003) diluted 1:50 in 1% (w/v) BSA in PBS. After washing with PBS, the signal was revealed with Alexa Fluor 488-labeled anti-rabbit IgG (Molecular Probes, Eugene, OR, USA) diluted 1:25 in 1% (w/v) BSA in PBS for 45 min in darkness. Finally, sections were counterstained with 1 mg/ml 4', 6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) for 10 min, mounted in Mowiol^� 4-88 (Sigma-Aldrich-Merck, Darmstad, Germany) and imaged under the confocal microscope (Leica TCS-SP5-AOBS). Laser excitation (488-nm argon laser) and sample emission capture settings (�63 oil immersion objective) were the same in all immunofluorescence preparations of each antibody. Controls were performed by omitting the primary antibody.

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.

MtCAS31 (full-length) and MtPIP2;7 (full-length) were expressed in the E. coli Rosseta strain using the pET-30a vector, purified and used to produce anti-MtCAS31 and anti-MtPIP2;7, respectively. The antibodies were produced by Beijing Huada Protein (http://proteomics.biogo.net/). The following antibodies were used: anti-FLAG (Sigma, F1804), anti-MYC (Sigma, M4439), anti-HA (Sigma, R3663), anti-GFP (Abmart, M20004S), anti-ATG8 (Abcam, ab4753), anti-His (Proteintech, 66,005–1-Ig), anti-GST (Proteintech, 66,001–2-Ig), anti-H⁺-ATPase (Agrisera, AS07-260), anti-Histone 3 (Agrisera, AS16-3968), anti-cFBPase (Agrisera, AS04-043), and anti-NPTII (Abcam, ab60018), anti-Actin (CWBIO, CW0264).