Cy3 conjugated goat anti rabbit igg

After dehydration and a blocking treatment, the lung tissue sections were incubated overnight at 4 °C with primary antibodies (TPSAB1 (1:200, 13343-1-AP, Proteintech, Wuhan, China), PAR2 (1:200, ab180953, Abcam, London, UK), GM-CSF (1:200, 17762-1-AP, Proteintech, Wuhan, China), IL-33 (1:200, 66235-1-Ig, Proteintech, Wuhan, China), and TSLP (1:200, ab47943, Abcam, London, UK)). The next day, the sections were treated with a corresponding fluorescently-labeled secondary antibody (Cy3 conjugated goat anti-rabbit IgG (1:300, GB21303, Servicebio, Wuhan, China) or Cy3 conjugated goat anti-mouse IgG (1:300, GB21301, Servicebio)) for 1 h at 25 °C, and were subsequently mounted using a VECTASHIELD mounting medium containing DAPI. Fluorescence images were acquired using the Nikon Eclipse C1 and Nikon DS-U3 systems (Nikon, Tokyo, Japan).

The mice with average tumor volume of about 100 mm³ were randomly allocated to 7 treatment groups (biological replicates n = 5 for each group), which were PBS, BPY, BPY@HSA, PBS + L, BPY + L, BPY-HSA + L, BPY@HSA + L. The tumor volume and body weight of each mouse was measured every two days to evaluate the tumor treatment efficacy. The dose of each formulation was 20 mg/kg (counted as BPY-Mal₂) for tail vein administration, and the laser irradiation (1 W/cm², 10 min) was performed at 24 h after administration. During the PTT processes, the mice were anesthetized by isoflurane and the temperatures of the irradiated sites were monitored by the thermal imaging camera (auto-capture mode with 30 s intervals). The tumors of represented mice were harvested for H&E staining, TUNEL and Ki67 (Anti-Ki67 Rabbit pAb, Servicebio, GB111499, dilution 1:500; Cy3-conjugated Goat Anti-Rabbit IgG, Servicebio, GB21303, dilution 1:300) analysis to evaluate the tumor inhibition effect 24 h after the first laser treatments (or 48 h after the first administration for groups without laser treatments). At the end of the treatments, the blood samples of each mouse were collected for blood routine analysis and blood biochemistry determination, and the major organs of represented mice were harvested for H&E staining to evaluate the safety of our formulations.

Human renal mesangial cells were cultured onto cover slips in 6‐well plates and then exposed as previously described. At the end of exposure, the cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature. Slides were washed in PBS for three times, the target preparation was defined using a liquid blocker pen. Subsequently, the target area was incubated with 3% BSA at room temperature for 30 minutes, and then with primary antibodies FN (ab2413; Abcam) and Col IV (ab6586; Abcam), overnight at 4°C, in a wet box. After three washing steps with PBS (pH 7.4), cell climbing slides were incubated with secondary antibodies, FITC conjugated goat anti‐rabbit IgG (GB22303; Servicebio) or Cy3 conjugated goat anti‐rabbit IgG (GB21303; Servicebio), at room temperature for 50 minutes. and then cells were stained with 0.2 mg/mL DAPI and analysed using a Zeiss LSM 880 confocal microscope (Carl Zeiss).

First, the heart sections were washed with PBS, and the primary antibody was diluted in FACS buffer and added into a hydrated chamber (anti-CD19 rabbit pAb [Servicebio]) overnight at 4 °C. Afterwards, sections were washed with PBS and stained with secondary antibody diluted in PBS for 1 h at 4 °C (Cy3 conjugated goat anti-rabbit IgG [Servicebio]). Sections were subsequently washed with PBS and incubated with DAPI solution for 10 min at room temperature. The sections were washed again with PBS, and then spontaneous fluorescence quenching reagent was added and the sections were incubated for 5 min. Finally, the sections were washed in running tap water for 10 min. Immunofluorescence images were obtained under a microscope (Olympus) at 400 times magnification and analysed with Image-Pro Plus 6.0 software.

Prior to transfection, cells were plated in a 12-well plate and grown to 50 ~ 60% confluence. Lipofectamine 3000 (Thermo) was used according to the manufacturer’s protocol. HA-TOX4 and GFP-TRIM65 plasmids were transfected into IEC-6 cells. 48 h later, IEC-6 cells were rinsed three times with PBS, fixed with 4% paraformaldehyde for 30 min at room temperature, and permeabilized in 0.1% Triton X-100 for 20 min. The cells were then incubated in 1% BSA in PBS for 1 h to prevent non-specific antibody binding. The cells were incubated with HA primary antibodies at 4 °C overnight, washed three times with PBS, incubated with Cy3-conjugated goat anti-rabbit IgG (GB21303, Servicebio) at 37 °C for 1 h, and the nuclei were stained with DAPI. A Laser-scanning confocal microscope was used to capture immunofluorescence images.