Goat anti rabbit igg h l secondary antibody

The protein samples were electrophoresed via 12% SDS-PAGE and transferred onto nitrocellulose membranes (Millipore, Germany). After blocking with 5% skim milk, the membranes were incubated with the rabbit polyclonal anti-HSP70 (Agrisera AS0837, Sweden,1: 3000), Rabbit polyclonal Rubisco large subunit,form I and form II antibody (Agrisera AS03037, Sweden 1:10000) at 4 °C overnight, respectively. Subsequently, the membranes were washed with PBS five times and incubated with goat anti-rabbit IgG(H + L) secondary antibody (Thermo Fisher Scientific, USA) at a ratio of 1:5000 at room temperature for 2 h. Finally, the membranes were developed using an enhanced chemiluminescence kit (Santa Cruz, USA⁾ [22 (link)].

GelMA with photoinitiator, lithium phenyl‐2,4,6‐trimethylbenzoylphosphinate, was purchased from Intelligent Manufacturing Research Institute (IMRI, GM‐90, Suzhou, China). TNF‐α, ox‐LDL, and IL‐1β were purchased from Sigma‐Aldrich (St. Louis, USA). DAPI, HUVECs, human SMCs, and actin‐tracker green were purchased from Keygen Biotech Co., Ltd. (Nanjing, China). Paraformaldehyde (4% w/v), Triton X‐100, CCK‐8, and phosphate‐buffered saline (PBS) were purchased from Solarbio (Beijing, China). Mouse MCs were purchased from Kemao Biotechnology (Dongguan, China). Penicillin–streptomycin, Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), and endothelial basal medium (F‐12K) were purchased from Gibco (Grand Island, USA). Cell labeling kits (Qtracker 525 and 655), α‐SMA, VE‐Cad primary polyclonal antibody, and goat anti‐rabbit IgG (H+L) secondary antibody were obtained from Thermo Fisher (Waltham, USA).

Cells were fixed and permeabilized with cold methanol:acetone (1:1) for 10 min at 4 °C according to our previous method²⁹ . In brief, cells were washed with 1× PBS and incubated in a blocking buffer (5% goat serum, Seracare Life Sciences Inc, 55600007) at room temperature for 30 min. Cells were then incubated with a primary antibody (monoclonal rabbit anti-SARS-CoV-2 N antibody, GeneTex, GTX135357) in 1× PBS (1:1000) overnight at 4 °C. The following day, cells were washed three times with 1× PBS and incubated with a secondary antibody (Goat anti-Rabbit IgG (H + L) secondary antibody, FITC (Thermo Fisher, 65-6111)) in 1× PBS (1:200) for 1 h at 37 °C. Then cells were washed three times with 1× PBS and incubated with DAPI (300 nM) (Thermo Fisher Scientific, D1306) for 5 min at room temperature. Images were acquired using a fluorescence microscope (ECHO, Revolve).
To measure the frequency of infected cells, randomly-selected areas were imaged. Each treatment had three replicates. The FITC-positive cells and DAPI-positive cells were quantified using CellProfiler software (4.2.1)²⁹ . The same threshold value was applied to the images of each area.
Quantification of the western blots was carried out with Image J software (1.52q). β-actin/GAPDH were used as loading controls.

After trypsin digestion, PBS washing, and centrifugation, total proteins from transfected cells were extracted using radioimmunoprecipitation assay buffer reagent (Solarbio). A BCA Protein Quantification Kit (Vazyme) was used to measure the total protein concentration of the clarified lysate. Cell lysates (20 μl) were loaded in each lane of 10% SDS‐PAGE gels and the protein samples were separated by constant voltage electrophoresis for 2.5 h (110 V). The separated proteins were transferred onto PVDF membranes by 1.5 h electrophoresis (Millipore). The membranes were washed in TBST and blocked with 5% fat‐free milk powder in TBST. Primary antibodies were added, and the membranes were incubated overnight at 4°C. After washing thrice with TBST (Tris‐buffered saline with Tween), the membranes were incubated with secondary antibodies at room temperature for 2 h. OD values of the targeted bands were analyzed using Gel‐Pro Analyzer software. Recombinant rabbit monoclonal antibodies against target genes were used as primary antibodies (Thermo Fisher). Goat anti‐rabbit IgG (H+L) secondary antibody was used as the secondary antibody (Thermo Fisher).