Gelatin was fluorescently labeled with Alexa-405-NHS ester and 35 mm glass bottom dishes (MatTek Corporation) were coated with Alexa-405-gelatin as previously described31 (link). 400,000 (4T1/67NR) or 300,000 (MDA-MB-231) cells were plated per dish and cells were fixed 18 h later with 4% paraformaldehyde (Alfa Aesar) for 10 min, permeabilized with 0.1% Triton X-100 (Calbiochem) for 5 min, blocked with 1% FBS/1% BSA (Sigma-Aldrich) in PBS (Gibco) for 3 h, incubated with anti Tks5 antibody (Millipore, MABT336) for 2 h, then with secondary antibody and Alexa Fluor 633 Phalloidin (Invitrogen) for 1 h.
Samples were imaged on a laser scanning confocal microscope (FV1200, Olympus) using a 60X objective (UPLSAPO60XS, 1.35 NA, Olympus). Stacks were collected at 1 µm z-step. To quantify matrix degradation, images were processed using a custom macro in Fiji. Briefly, slices from the stack were z-projected using the Max Intensity method, followed by thresholding of the signal in the gelatin channel, using the Automatic Threshold algorithm, and measuring the area of degradation spots using the Particle Analysis tool. To account for the differences in the cell density across fields of view, the total area of degradation in a field of view was divided by the total number of cell present in this field of view. Cells were counted using the F-actin staining.
Samples were imaged on a laser scanning confocal microscope (FV1200, Olympus) using a 60X objective (UPLSAPO60XS, 1.35 NA, Olympus). Stacks were collected at 1 µm z-step. To quantify matrix degradation, images were processed using a custom macro in Fiji. Briefly, slices from the stack were z-projected using the Max Intensity method, followed by thresholding of the signal in the gelatin channel, using the Automatic Threshold algorithm, and measuring the area of degradation spots using the Particle Analysis tool. To account for the differences in the cell density across fields of view, the total area of degradation in a field of view was divided by the total number of cell present in this field of view. Cells were counted using the F-actin staining.