Alexa fluor 555 anti mouse igg

H1299 or A375 cells were allowed to adhere to a cover slip overnight in DMEM complete media and grown until they reached approximately 75% confluency on the cover slip surface. After a 30 min treatment with 20 μm peptides, TAT (used as a negative control), TAT‐E1A‐WT, and Pep1‐E1A‐WT, cells were fixed for 10 min using 100% methanol, and permeabilized for 1 min in 100% acetone. Each sample was stained with an anti‐FLAG antibody and detected using an Alexa Fluor 555 anti‐mouse IgG (4409S; Cell Signaling Technology, Danvers, MA, USA). DAPI staining was used to visualize the nucleus.

We captured cells on the cover glass and fixed them with 4% paraformaldehyde (PFA, Invitrogen, CA, USA) for 15 min. The cells were incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies that were fluorescently labelled. The primary antibodies used were as follows: rabbit anti-VE-cadherin (1:250; cat. no. 2500, Cell Signaling Technology, MA, USA), rabbit anti-Iba-1 (1:250; cat. no. ab178846, Abcam, MA, USA), and mouse anti-Thrombospondin 1 (1:250; cat. no. ab1823, Abcam, MA, USA). The secondary antibodies used were Alexa Fluor 488 anti-rabbit IgG (1:500; cat. no. 4412, Cell Signaling Technology, MA, USA) or Alexa Fluor 555 anti-mouse IgG (1:500; cat. no. 4409, Cell Signaling Technology, MA, USA). Afterward, the Nuclei were stained with DAPI for 5 min in the dark. All images were captured using a confocal microscope (Zeiss LSM 980 with Airyscan 2, Oberkochen, Germany) or a fluorescence microscope (Zeiss Axio Imager Z1, Oberkochen, Germany).

Bel7404 and HepG2 cells were plated in 35 mm confocal culture dishes for 24 h. On the following day, the cells were treated as previously described [24 (link)]. Primary antibodies against SCUBE3 (ab189955, Abcam,1:50) and TGFβIIR (sc-17799, Santa cruz,1:50) were used at 1:50 dilution. After washing, the cells were then incubated with fluorescence-labelled secondary antibodies Alexa-Fluor 555 anti-mouse IgG and anti-rabbit IgG (1:200 dilution, Cell Signaling Technology, Danvers, MA, USA). Then, cells were washed and stained with DAPI. Finally, images of cells positive for SCUBE3 and TGFβIIR were captured using a confocal microscope (Nikon Corporation, Tokyo, Japan).