Prolong diamant antifade mountant with dapi

To analyze localization of Lpd, glioblastoma cells were stained with Lamellipodin pab 3917, AlexaFluor488anti-rabbit (A32731, Life Technologies, Carlsbad, CA, USA), AlexaFluor633 Phalloidin (A22284, Life Technologies) and mounted using ProLong Diamant Antifade Mountant with DAPI (Thermo Fisher Scientific) as described [9 (link)]. Images were acquired using LSM980 Airyscan 2 (Carl Zeiss Microscopy).

To analyze the endogenous localization of ADAM10 (1:100, AB19026, Millipore) and CX3CL1 (1:100, ab25088, Abcam), heart tissue sections (mid-ventricular short axis sections, 4% PFA fixed, paraffin embedded, 3 µm) and indicated cells were stained with specific antibodies and counterstained with CD31 (1:100, 553370, BD Biosciences), CD45 (1:100, 555480, BD Biosciences), cTnI (1:100, MAB3150, Millipore), cTnT (1:100, ab45932, Abcam) or Vimentin (1:100, 550513, BD Biosciences) where indicated. Alexa Fluor 488-coupled goat anti-rabbit (1:200, A27034, ThermoFisher Scientific), Alexa Fluor 546-couppled goat anti-mouse (1:200, A-11003, ThermoFisher Scientific) or CF 594-coupled goat anti-rat (1:200, SAB4600323, Sigma-Aldrich) and ProLong Diamant Antifade Mountant with DAPI (P36962, ThermoFisher Scientific) were used as secondary antibodies and for mounting, respectively. Image acquisition was performed using a Keyence BZ-X710 All-in-One Fluorescence Microscope (Keyence) with the BZ-X Viewer v1.03.00.05 software (Keyence). The cellular distribution of ADAM10 and CX3CL1 was assessed using CellProfiler 4⁴⁶ . Cardiac-TnT, CD31, CD45 and Vimentin positive cells were analyzed for ADAM10 expression. For each mouse at least 1000 cells/staining were analyzed. Cardiomyocyte cross-sectional diameter was determined using Fiji. One hundred cells/mouse were analyzed.