Col 1 antibody

Silicone wells attached to Silane-Prep slides (Sigma) were coated with 10 μl of TMG (2 mg/ml), human Col I (2 mg/ml, Advanced Biomatrix), or Matrigel (5 mg/ml, Corning). The gels were polymerized in a 37 °C incubator for 1 h. MM231 cells were seeded on the matrix (2000 cells/well) and cultured for indicated hours (37 °C, 5% CO₂). The cells were then fixed with 4% paraformaldehyde, washed with 1× PBS, permeabilized with 0.1% Triton-X 100, blocked with 3% BSA in PBS, and incubated with Col I antibody (Abcam) and Alexa Fluor probe-conjugated phalloidin (ThermoFisher) in dark at 4 °C overnight. The cells were washed with 0.1% Triton-X 100 and incubated with Alexa Fluor 488-conjugated secondary antibody (ThermoFisher) against Col I for 1 h in dark at room temperature, followed by washing with 0.1% Triton-X 100 and Hoechst (TOCRIS) staining of cell nucleus. Coverslips were mounted on top of the IF-stained samples using FluorSave medium (MilliporeSigma). The slides were stored in dark at 4 °C overnight and staining imaged using a Zeiss Imager M2 upright epifluorescence microscope. The protrusions of 10 randomly selected cells cultured on individual matrix-coated slides were counted, and the collective counts from three biological repeats were compiled and compared using Student’s t test.

Fish collagen sponge (which was extracted by acid-enzyme combination from tilapia skin (Pal et al., 2016 (link))), Bovine collagen sponge (Wuxi Beidi Bioengineering Co., LTD.), SD rat (Jinan Peng Yue Animal Breeding Center), Immunohistochemical kit (Beijing Solaibao Technology Co., LTD.), FGF antibody, VEGF antibody, Col I antibody (Abcam), Col III antibody (Amyjet), FITC (Thermo), CCK-8 kit (Beijing Solaibo Technology Co., LTD.), Live cell/dead cell double staining kit (Calcein-AM/PI), L929 (mouse fibroblast, Qingqi (Shanghai) Biotechnology Development Co., LTD.), Roswell Park Memorial Institute (RPMI) 1640 (Thermo), Glutaraldehyde (Maclin), Hematoxylin-Eosin (HE) Stain Kit (Beijing Solaibao Technology Co., LTD.), Masson’s Trichrome Stain Kit (Beijing Solaibao Technology Co., LTD.).