Jpk bioafm nanowizard 3

Cell nuclei of excitatory neurons in the visual cortex of Nex‐Cre;SUN1‐GFP mice were attached to the Poly‐D‐Lysine (PDL)‐coated Petri dishes (World Precision Instrument, FD5040‐100) and subjected to AFM‐based mechanical testing (Maki et al., 2016 (link)). In this study, we utilized JPK BioAFM NanoWizard 3 (Bruker Nano GmbH), which was mounted on an epi‐fluorescence microscope (IX81; Evident). The spring constants of AFM cantilevers (qp‐BioAC CB2; Nanoworld AG) were calibrated based on the thermal noise method (Butt & Jaschke, 1995 ). Cell nuclei of excitatory neurons were fluorescently identified with GFP. AFM mechanical testing for the GFP‐positive nuclei was conducted with the following settings; the piezo displacement speed of 6 μm/s and the sampling rate of 4000 Hz. Based on the indentation force (F) versus depth curve obtained by mechanical testing, we estimated slope (nN/μm) by linear regression for the sample points within a force range (500 pN ≤ F ≤ 1000 pN), as previously described (Ichijo et al., 2022 (link); Maki et al., 2015 ).

E11.5 mouse embryos were extracted and snap-frozen in O.C.T. compound with liquid nitrogen. Those embryos were then serially cryo-sectioned by a microtome with a thickness of 20 μm. To identify nascent cartilage and muscle regions for AFM measurement, we performed immunofluorescence staining (cartilage: Sox9, muscle: MyoD) and in situ mRNA hybridization (cartilage: Col2a1, muscle: MyoD) for one-before and one-after cryo-sections, respectively. Finally, those staining results for those neighboring cryo-sections were collated with AFM results. For AFM-based tissue indentation experiment, the cryo-section was thawed and the nuclei were immediately stained with Hoechst 33342 in PBS(-). In this study, AFM system (JPK BioAFM NanoWizard 3; Bruker Nano GmbH., IX81; Olympus Co.) was used and AFM cantilevers (TL-CONT; spring constant 0.2 N/m; NANOSENSORS) were modified with glass beads (15 μm-diameter; Polysciences. Inc.). Spring constant of the AFM cantilever was calibrated with a thermal noise method. Piezo displacement speed was set to be 3 μm/s. As a result of tissue indentation, force (F) versus indentation depth (h) curves were obtained and slope (nN/μm) was evaluated for each curve by linear regression. Sample points within the force range of 750 pN≤F≤ 1000 pN were used for the evaluation. After AFM, the sample was immunostained for Sox9 and MyoD.