Anti furin antibody

The selected tissues were fixed in 10% neutral formalin solution and embedded in paraffin. The paraffin sections (4 μm in thickness) were deparaffinized in xylene, rehydrated in a graded series of alcohol. Human lung bronchi tissue sections were used as positive control of ACE2 and Furin expression. The tissue sections were then incubated in citrate buffer (pH 6.0) for 5 min at 120°C, and the endogenous peroxidase was blocked by 0.3% H₂O₂ for 10 min. Subsequently, the tissue sections were dried and incubated with Rabbit Anti-ACE2 antibody (cat. no. ab15348, Abcam, Cambridge, CB, UK) antibody, Anti-Furin antibody (cat. no. bs-13228R) overnight at 4°C. The negative control sections were incubated with 1% rabbit serum containing phosphate buffered saline (PBS) instead of the primary antibody. The next day, the tissue sections were incubated with the horseradish peroxidase (HRP) (Goat Anti-Rabbit IgG H&L)-conjugated secondary antibody for 1 h at room temperature. Peroxidase activity was developed by using 3,3′-diaminobenzidine (DAB) for 8 min and counterstained with hematoxylin. A qualified pathologist analyzed the staining. The ACE2 and Furin staining intensities were semiquantitatively graded according to the previously described method (30 (link)).

Breast cancer cells were fixed with 4% paraformaldehyde and then incubated in immunostaining blocking buffer (Beyotime P0102) for 1 h to permeabilize the cells and block nonspecific protein–protein interactions. For colocalization, cells were then incubated with anti‐Plac1 antibody (1 : 100; Abcam) and anti‐Furin antibody (1 : 100; Abcam), followed by Alexa Fluor^® 488 and Alexa Fluor^® 594‐secondary antibody, respectively (Yeasen, Shanghai China). Nuclei were detected using DAPI staining. For NICD nuclear import, cells were then incubated with anti‐NICD antibody (1 : 100; Abcam) followed by Alexa Fluor^® 594‐secondary antibody (Yeasen). Proteins were then detected using confocal microscopy.