Hrp labeled goat anti rabbit secondary antibody

Polylysine-coated coverslips were used, and the treated cells were grown on a coverslip and fixed with 4% paraformaldehyde solution for 15 min. After the formaldehyde was discarded, 0.1% Triton X-100 was added followed by incubation for 20 min. The solution was then removed, and the cells were washed with 0.01 M PBS 3 times for 5 min each time. After the PBS was discarded, 3% H₂O₂ was added followed by incubation for 15 min, and washing with 0.01 M PBS 3 times for 5 min each time. The PBS was discarded, and serum was added to block for 15 min. The serum was then removed, and anti-Bcl-2 antibody (diluted 1:100; WL01556; Wanleibio) was added followed by incubation overnight at 4°C. The primary antibody was discarded, and the cells were washed with 0.01 M PBS 3 times for 5 min each time. After the PBS was discarded, HRP-labeled goat anti-rabbit secondary antibody (diluted 1:100 with PBS; g1213, Servicebio, Wuhan, China) was added to completely cover the cells and incubated at 37°C for 60 min. The secondary antibody was discarded, and the cells were washed with 0.01 M PBS 3 times for 5 min each time. DAB was added for color development, and then the cells were counterstained with hematoxylin (g1004-100; Servicebio). The cells were mounted and observed under a microscope. Photos were taken, and optical density was analyzed using Image-Pro Plus software.

The abdominal adipose and liver tissue of the mice were fixed in 4% paraformaldehyde for 24 h. Paraffin-embedded 5 μm sections were cut and mounted on slides. Adipose tissue and liver tissue sections were stained with hematoxylin and eosin for morphological analysis. Immunohistochemical staining of the liver tissue sections were immersed in boiling citric acid antigen retrieval buffer for antigen retrieval. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in distilled water. The slides were incubated in COX-2 monoclonal antibody (Servicebio, China; dilution 1 : 1000), followed by HRP-labeled goat anti-rabbit secondary antibody (Servicebio, China; dilution 1 : 200). The final DAB visualized positive expression was brownish yellow, and the hematoxylin-stained nucleus was blue. The stained slides were scanned by a Pannoramic SCAN system (3DHISTECH, Hungary) to obtain representative images.

T4 polynucleotide kinase and T4 DNA ligase were purchased from Takara Biotech, Co., Ltd. (Dalian, China). DMT enzyme was obtained from TransGen Biotech Co., Ltd. (Beijing, China). Paclitaxel (>97% purity) was purchased from Energy Chemical (Shanghai, China). Anti-transferrin receptor rabbit pAb and anti-neuropilin-1 rabbit mAb were purchased from Beyotime Biotechnology (Shanghai, China). Anti-GAPDH rabbit pAb and HRP-labeled goat anti-rabbit secondary antibody were purchased from Servicebio (Wuhan, China). MTT was purchased from Guangzhou Saiguo Biotech Co., Ltd (Guangzhou, China). Penicillin-streptomycin solution (100×) and 0.25% trypsin-EDTA (1×) solution were purchased from Biosharp Life Sciences. Fetal bovine serum (FBS) was purchased from Zhejiang Tianhang Biotechnology Co., Ltd. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from HyClone. Fluorescein isothiocyanate (FITC) was purchased from Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China). DAPI was purchased from Nanjing KeyGen Biotech. Co., Ltd. (Nanjing, China). All cell lines were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China).