Zen 3.0 blue

For quantitative analysis, confocal images were captured using a 63 × objective lens with an Airyscan under constant viewing settings. To compare glial contacts of OPN deposits in the hippocampal CA1 region, we used 78.01 × 78.01 μm images of 6.8 μm Z stacks, with 0.3 μm optical sections, from eight different animals. The perimeter of each OPN deposit and Iba1 or GFAP-labeled perimeter were measured using Zen 3.0 blue (Carl Zeiss).
Statistical significance was determined through the Student's t-test, with considering differences as significant when P values were < 0.05. The number of animals assessed, and the P values are indicated in figure legends and the graphs. All statistical analyses were conducted using the Prism 7 software (GraphPad Software Inc., San Diego, CA, USA).

3 × 10⁴ MC3T3-E1 cells were seeded onto the scaffolds and cultured for 3 days in GM. Then, actin and cell nuclei were stained using Alexa Fluor 568 Phalloidin (ThermoFisher, A12380) and DAPI (ThermoFisher, 62247) by following the manufacturer’s protocol. Briefly, after washing with PBS, samples were fixed in 4% paraformaldehyde (PFA) in PBS for 15 min. After washing with PBS, the cells were permeabilized using 0.1% Triton X-100 in PBS for 15 min, followed by incubation in blocking buffer (0.1% Triton X-100, 1% BSA in PBS) for 45 min. For immunofluorescence staining of actin, samples were stained with fluorescent phalloidin staining solution for 60 min and rinsed three times with PBS. For DAPI staining, the samples were stained with the DAPI solution for 10 min and rinsed five times with PBS to remove excess staining solution. Finally, the samples were visualized with CLSM, and actin length, cell area, and fluorescence intensity were measured and analyzed using ZEN software (Zen 3.0 Blue, Zeiss).

PLA images were processed using ZEN 3.0 (blue edition) (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) software. The images of each z-stack were combined into an orthogonal projection. For all images, the background was reduced equally by adjusting the intensity values. PLA signal density heatmaps were generated using Fiji (ImageJ 1.53c) [41 (link)], applying the Lookup Table “Red Hot” and a Gaussian Blur filter with a Sigma radius of 20. For better orientation, the heatmap image was overlayed onto a high-background image showing the nuclei generated by using the “remove outliers” function of Fiji.