Hrp conjugated goat anti rabbit secondary antibody

The total protein was separated from the transformed tobacco leaves through the adoption of the isolation buffer, and the protein concentration was measured using the blood alcohol concentration method. The protein samples were boiled for 12 min after mixing with the buffer. The lysates were split by SDS-PAGE and checked by immunoblotting against the rabbit anti-GFP antibody (Abcam, ab290, Cambridge, MA, USA) for GFP-MtTCP4. As a loading control, actin was detected with the rabbit anti-Actin antibody (Abcam, ab197345, Cambridge, MA, USA). The HRP-conjugated goat anti-rabbit secondary antibody (Beyotime, A0216, Beijing, China) was adopted for anti-GFP or anti-actin immunoblotting.

The anti-TAGAP (1:100) and anti-CD8/CD4 (1:100) were purchased from Solarbio-Beijing and ABclonal.The goat anti-rabbit secondary antibody was purchased from Beyotime Institute of Biotechnology.
Fixed tumor tissues were dehydrated, embedded in paraffin, and sliced into 4 μm sections. The sections were deparaffinized in two xylene baths for 20 minutes each, followed by hydration in a series of graded ethanol baths for 5 minutes each. Antigen retrieval was performed using EDTA antigen retrieval solution for 20 minutes. After PBS washing, the sections were incubated with a peroxidase-blocking reagent at room temperature for 10 minutes to block endogenous peroxidase activity. The sections were then blocked with normal goat serum for 30 minutes. CD4 antibody (ABCAM, USA) was added to the tissue sections and incubated overnight at 4°C. Afterward, the sections were incubated with HRP-conjugated goat anti-rabbit secondary antibody (Beyotime, China) at 37°C for 30 minutes, followed by 5-minute DAB staining. After PBS washing, the sections were counterstained with hematoxylin, dehydrated in graded ethanol and xylene, and mounted. Photographic documentation and analysis of the staining results were performed using an upright light microscope (Leica DM 4000B, Germany).

Exosomal protein content was measured using a BCA assay. Total protein (40 μg) was electrophoresed using 10% SDS–PAGE, and transferred to PVDF membranes (Millipore, USA). Membranes were blocked for 1 h at RT with 5% non-fat milk. Then membranes were incubated overnight at 4 °C with the primary antibody (rabbit anti-CD29, 1:1000, Proteintech or mouse anti-CD63, 1:1000, Abcam) diluted in TBST with 5% BSA. Membranes were washed with TBST, followed by incubation for 1 h at room temperature with either horseradish-peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies or HRP-conjugated goat anti-mouse (1:1000, Beyotime). The bands were visualized using ECL detection.