Metal enhanced dab substrate kit

Deparaffinated sections of the aorta were prepared by heating the sample for 20 min, cooling the sample at room temperature, and then washing the sample 3 times with 0.01 M PBS (pH 7.2–7.6) for 3 min. Endogenous peroxidase activity was blocked by incubating the sections in 1.0% periodic acid for 10 min and then washing them 3 times with 0.01 M PBS for 3 min. The sections were incubated with rabbit anti-mouse iNOS at 4 ℃ overnight. After the sections were washed 3 times with PBS for 5 min, they were incubated with anti-rabbit IgG-HRP at 37 ℃ for 30 min. They were then washed 3 times with PBS for 5 min, and further treated with Metal Enhanced DAB Substrate Kit (Solarbio) at room temperature for 1–5 min. Following hematoxylin staining, alcoholic dehydration, xylene treatment and application of a neutral resin sealing sheet, the sections were visualized using a DAB kit (ZSGB-BIO). The antibodies information was showed in Table 2.

To prepare the animal tissue samples, we embedded the formalin‐fixed tissue samples in paraffin, cut them into 4 μm sections and placed them on slides. For immunohistochemical staining, the slides were dewaxed in xylene and rehydrated with graded alcohol. Then, the slides were incubated in 3% hydrogen peroxide to impede endogenous peroxidase activity. The slides were boiled for 30 min in 10 mM sodium citrate (pH 6.0) for antigen retrieval, blocked with 5% normal goat serum for 15 min and then incubated with the indicated antibodies overnight at 4°C in a humid room. The next day, the slides were incubated with the secondary antibody for 1 h at room temperature after the PBS washing process. A metal enhanced DAB substrate kit (Solarbio Life Sciences, Peking, China) was used to detect immunoreactivity. The antibody staining intensity in tissue sections was quantitatively assessed by an IHC Profiler¹⁴ using ImageJ (an open‐source plugin for quantitatively evaluating and automatically scoring immunohistochemistry images of human tissue samples). The IHC profiler uses the average gray value of positive cells (staining intensity) and the percentage of positive area (stained area) as IHC measurement indicators and ultimately gives four scores: high positive (3+), positive (2+), low positive (1+) and negative (0).

Samples were embedded in paraffin for long-term storage. Tissue sections were prepared using a graded alcohol series. The slides were blocked using 0.3% hydrogen peroxide for 15 min and then boiled in the aminomethane-EDTA buffer (pH 8.0) for another 30 min. 10% normal goat serum was used to block non-target antigens for 20 min. Slides were incubated with the primary antibody overnight at 4 °C and horseradish peroxidase (Copenhagen, Denmark). The staining was visualized using Metal Enhanced DAB Substrate Kit (Solarbio) according to the manufacture’s protocol.

After injection with immune elicitors, tobacco leaf samples were collected from the injection site, and total protein was extracted using the plant protein extraction kit. Western blot analysis was performed following the method described by Chen et al. [35 (link)]. Specifically, anti-BAX, anti-HA and anti-FLAG mouse monoclonal antibodies (TransGen, Beijing, China) were used to detect BAX protein, RBP-1 protein, and RsVAP and RsVAP^ΔSP, respectively. Goat anti-mouse IgG and HRP conjugate (TransGen, Beijing, China) were used as secondary antibodies for all Western blot analyses. Metal enhanced DAB substrate kit (Solarbio, Beijing, China) was used for protein staining.

Spore coat proteins were extracted from the purified spores using an SDS-DTT extraction buffer, as described in detail elsewhere [30 ]. For western blot analysis, 50 µg of the extracted proteins were fractionated on 10% SDS-PAGE, electro-transferred to polyvinylidene difluoride (PVDF) membrane (Pall, Hercules, CA, USA) using minitransfer blot (Bio-Rad, Hercules, CA, USA). The Western blot assays were performed using previously described protocols [25 (link)]. Reactive bands were detected with enhanced chemiluminescence (ECL) reagent (Sigma-Aldrich, St.Louis, Missouri, USA) and exposed to X-film as previously described [25 (link)]. For dot blot analysis, 2 µL of 10-fold dilution of the coat proteins with a defined amount was spotted on the FVDF membrane. After treatment, as described in Western blot analysis, the filter was stained with Metal Enhanced DAB Substrate kit (Solarbio, Beijing, China). Images were analyzed densitometrically using Image J.