Dab solution

The MDSCs were fixed with an ice-cold 4% paraformaldehyde, incubated with 3% H2O2 for 30 min., and blocked with goat serum. The cells were then incubated with neuron-specific enolase (NSE) (1 : 100, sc-51880) overnight at 4°C, followed by a goat anti-mouse IgG (1 : 100; sc-2005, Santa Cruz Biotechnology, USA). Next, cells were incubated with DAB solution (D3939, Sigma, USA).

For testing the ROS burst in the leaves by DAB (3,3′-diaminobenzidine) staining, detached leaf samples were collected at 2 d after infiltration of A. tumefaciens strains as previously described (Dong et al., 2011 (link)). Leaves were stained with 1 mg ml^–1 DAB solution (Sigma-Aldrich, USA) at 25 °C for 6 h, then decolorized with boiling alcohol, and kept in 30% glycerin. To determine the role of Al6 in the ROS burst in response to flg22, a luminol-based assay was adopted as previously described (Trujillo, 2016 (link)). After the instantaneous expression of GFP and Al6 in N. benthamiana for 36 h, leaf disks were washed with water and kept overnight in the dark at room temperature. Then, the leaf disks were stained in test buffer containing luminol (Sigma-Aldrich, USA), horseradish peroxidase (Sigma-Aldrich, USA), and 1 μM flg22 (GenScript, China), and luminescence detection was conducted for at least 30 min in the microplate reader (BioTek, China).

Mice were transcardially perfused with 4% paraformaldehyde (PFA) and postfixed for an additional 24 h. Brains were then transferred to PBS and immersed into 4% agarose before sectioning. 3,3′-Diaminobenzidine (DAB) staining was carried out as before (Engel et al., 2006 (link)). Slices were treated with 1% H₂O₂ for 45 min to inactivate endogenous peroxidases. This was followed by an incubation with blocking solution (10 ml: 8.3 ml 1× PBS, 1 ml 10% BSA, 0.5 ml 5% FBS, 0.2 ml Triton-X-100) for 1 h and primary antibody (AT8, 1:100, Invitrogen, CA, USA) overnight at 4°C. On the following day, tissue sections were washed 3× with PBS for 10 min each and incubated with Vectastain kit (Vector Laboratories, Burlingame, CA, USA; one drop of biotinylated antibody and three drops of horse/donkey serum were mixed in 10 ml of 1% BSA-PBS). This solution was added to the tissue and incubated for 90 min at room temperature. Three washes were performed with PBS for 10 min followed by a 90 min incubation with Avidin (ABC) peroxidase complex at room temperature (two drops of reagent A and reagent B in 10 ml of 1% BSA-PBS). Slices were washed with PBS for 20 min and immersed in DAB solution (Sigma-Aldrich, Arklow, Ireland) for approximately 10 min. Then, slices were mounted using FluorSave^TM. Staining was examined under a light microscope.

H₂O₂ accumulation in plant tissue was examined by staining with DAB as described previously (Xiao et al., 2003 (link)). Leaf pieces were infiltrated in DAB solution (Sigma) (1 mg/ml, pH 3.8) and shaken at room temperature for 12 h in the light. The leaf pieces were then destained with 95% ethanol. The cleared leaves were transferred to 50% glycerol. A microscope (Olympus) was used to take photographs. The accumulation of ROS was evaluated by ImageJ.

The method was basically adapted from a previously published protocol [27 (link)]. First, 30 µg of total protein extract was loaded on three identical 10% native polyacrylamide gels. Afterwards, electrophoresis for 16 h at 70 V and 4 °C was performed. Gel 1 was stained with Coomassie as a loading control. Gel 2 was used for peroxidase activity staining: the gel was washed three times for 10 min in PBS (pH 7.4) and then incubated in 30 mL DAB solution (30 mg diaminobenzidine; Sigma-Aldrich, St. Louis, MO, USA; D8001) dissolved in 30 mL PBS (pH 7.4) and 3 µL 30% hydrogen peroxide (Carl Roth, Karlsruhe, Germany; 8070.2) for 20 min under shaking. To stop the reaction, the gel was washed in H₂O twice. Gel 3 was used for catalase activity staining: the gel was washed with H₂O three times (10 min each) and subsequently incubated in 30 mL of H₂O with 3 µL of 30% hydrogen peroxide under shaking in the dark. The gel was washed with H₂O twice and was incubated for 10 min on a light table with 2 g FeCl₃ × 6 H₂O (Sigma-Aldrich, St. Louis, MO, USA; P742.1) in 10 mL H₂O and 1.2 g K₃[Fe(CN)₆] (Carl Roth, Karlsruhe, Germany; 244023) in 10 mL H₂O. Both solutions were mixed and immediately used. Afterward, the gel was washed with H₂O and documented by scanning.

Reactive oxygen species levels were tested by DAB (3, 3′-diaminobenzidine) staining. The candidate effectors were expressed in N. benthamiana leaves for 2 days, and immersed in 1 mg/ml DAB solution (Sigma-Aldrich, St. Louis, CA, United States) at 25°C for 6 h, then decolorized in industrial alcohol and preserved in 30% glycerol. Finally, photographs were taken at natural light.