Sc 81703

HEK and HaCaT were seeded at 1.0 × 10⁴ cells/well with a cover glass in a 12-well plate and cultured at 37 °C with 5% CO₂ for 24 h. The cells were fixed with 95% ethanol at RT for 10 min and then dried at RT. The cells were blocked with 10% FBS for 30 min at 37 °C. After being washed three times with PBS, the cells were incubated overnight with a rabbit anti-human OPN3 polyclonal antibody (AB_2837240, 1:50, Affinity Biosciences Ltd., Beijing, China) and a mouse anti-human pan-cytokeratin monoclonal antibody (sc-81703, 1:50, Santa Cruz Biotechnology, Inc., USA) at 4 °C. Following washing with PBS three times for 5 min, the cells were incubated for 45 min with Alexa Fluor 488-labeled goat anti-rabbit IgG (A0423; 1:50, Beyotime Biotechnology, Haimen, Jiangsu, China) or Cy3-labeled goat anti-mouse IgG (A0521; 1:50, Beyotime Biotechnology, Haimen, Jiangsu, China) in the dark at  37 °C. Finally, the cell nucleus was stained with DAPI (C0065, Solarbio, Beijing, China) for 10 min at RT. Fluorescent images were collected by fluorescence microscopy (Zeiss, Oberkochen, Germany).

The skin explants were fixed with neutral-buffered 10% formalin overnight, washed with PBS, and then embedded in paraffin. Sections were cut into 3-µm thick slices, deparaffinized in xylene, hydrated in a graded series of ethanol, and subjected to antigen retrieval by the microwave method with EDTA. The samples were blocked with 10% bovine serum albumin and 0.5% Tween-20 in PBS. The slides were incubated overnight with primary antibodies at 4 °C. Incubation with fluorescence-labeled secondary antibodies was performed for 30 min at 37 °C. The following primary and secondary labeled antibodies were used: rabbit polyclonal to OPN3 N-terminal (ab228748; Abcam) conjugated to Alexa Fluor 488-labeled goat anti-rabbit IgG (A0423; Beyotime); mouse anti-human pan-cytokeratin monoclonal antibody (sc-81703; Santa Cruz Biotechnology, Inc.) conjugated to Cy3-labeled goat anti-mouse IgG (A0521; Beyotime); rabbit anti-human Dync1i1 polyclonal antibody (AB_2846296, Affinity Biosciences, Ltd.) conjugated to an Alexa Fluor 488-labeled goat anti-rabbit IgG (A0423; Beyotime); and rabbit anti-human DCTN1 polyclonal antibody (AB_2838577, Affinity Biosciences, Ltd.) conjugated to Alexa Fluor 488-labeled goat anti-rabbit IgG (A0423; Beyotime). Nuclear staining was performed with DAPI. Fluorescent images were collected by fluorescence microscopy (Zeiss, Oberkochen, Germany).