Gold conjugated anti rabbit igg

Extracted tau filaments were deposited on glow-discharged 400 mesh formvar/carbon film-coated copper grids (EM Sciences CF400-Cu) for 40 s, blocked for 10 min with PBS + 0.1% gelatin, and incubated with primary antibody (1:50) in blocking buffer, essentially as described [20 (link)]. Primary antibodies were BR136 (raised against residues 244–257) [12 (link)], Anti-4R (raised against residues 275–291, with D279) [10 (link)], BR135 (raised against residues 323–335) [21 (link)], and TauC4 (raised against residues 354–369) [38 (link)]. Where stated, grids were incubated for 5 min with 0.4 mg/ml pronase (Sigma) in PBS at room temperature and washed with blocking buffer, prior to blocking. Following incubation with primary antibodies, grids were washed with blocking buffer and incubated with 10 nm gold-conjugated anti-rabbit IgG (Sigma) diluted 1:20 in blocking buffer. The grids were then washed with water and stained with 2% uranyl acetate for 40 s. Images were acquired at 11,000 × and 15,000 ×, with a defocus value of -1.4 μm with Gatan Orius SC200B or Gatan Ultrascan 1,000 CP CCD detectors using a Tecnai G2 Spirit at 120 kV. To distinguish non-specific background levels of isolated gold beads from specific labelling of the filaments, we only considered filaments with more than six gold beads to be positively labelled.

Ultrathin sections of grapevine leave were prepared from Lowicryl K₄M-embedded specimens, according to Peng’s method [46 (link)]. Ultrathin sections were transferred to a drop of 2% (w/v) bovine serum albumin (BSA) in tris-buffered saline (TBS) (pH 7.5) and blocked at room temperature for 1 h. Sections were then incubated for 1 h with the antiserum, which was diluted 100-fold in TBS buffer containing 1% (w/v) BSA. Subsequently, the sections were washed three times with TBS buffer for 10 min each. The washed sections were then placed on a drop of 10 nm gold-conjugated anti-rabbit IgG (Sigma-Aldrich) diluted 50-fold in TBS and incubated for 1 h. Samples were washed three times with TBS buffer and then two times with distilled water. Sections were then counterstained with 2% (w/v) uranyl acetate. Gold-labeled sections were examined under a JEOL (Tokyo, Japan) JEM 1220 transmission electron microscope. Rabbit pre-immune serum was used as a control.

Ultrathin sections (70 nm) of mature spores were prepared as previously described (60 (link)) and placed on nickel grids (Quantifoil, Beijing, China). After blocking with 10% (vol/vol) non-specific goat serum together with 5% (wt/vol) BSA in PBST at room temperature for 1 hour, the grids were incubated with STX 6 polyclonal antibody (1:50 dilution; ImmunoWay, Jiangsu, China) or the negative rabbit antiserum at room temperature for 1 hour. The grids were then incubated with gold-conjugated anti-rabbit IgG (1:30 dilution; Sigma-Aldrich, St. Louis, USA). Then, the samples were stained in 3% (wt/vol) uranyl acetate (Zhongjingkeyi Technology Co., Ltd., Beijing, China), followed by 1% (wt/vol) lead citrate (Zhongjingkeyi Technology Co., Ltd., Beijing, China). Samples were observed using the Hitachi HT7800 electron microscope (Hitachi, Tokyo, Japan) at 80 kV.