Imaris software bitplane

Confocal imaging of cerebrovasculture was carried out on a Leica SPE confocal microscope (Leica Microsystems) using a 10× and 20× objective. Adult zebrafish heterozygous for kcnj8 V65M, abcc9 G989E and abcc9 C1043Y and carrying the kdrl:GFP transgene were interbred, and genotype identified by Sanger sequencing post-imaging. Zebrafish at 5 dpf were anesthetized with 16 mg/ml tricaine and mounted in dorsal position in 0.25% agarose. 3D quantification of cerebral vasculature was carried out with Imaris software (Bitplane, Oxford Instruments). A Leica SPE confocal system (Leica Microsystems) was used to generate confocal stacks of approximately 250 μm with a slice interval of 1 μm under identical imaging conditions for all samples in an experiment. Stacks were aligned and reconstructed into a 3D volume using Imaris. The volume of the structure resembling the human circle of Willis was measured after eliminating any interfering vessels.

PANC‐1 cells were grown overnight on 35 mm confocal dishes and washed with PBS. First, cells were fixed in 4% formaldehyde solution in PBS for 10 min at room temperature and then permeabilized with 0.2% (v/v) Triton X‐100 (Sigma‐Aldrich) in PBS at room temperature. The cells were subsequently rinsed with PBS and incubated with Hoechst 33342 (1:2,000 dilution) for 10 min at room temperature. For mRNA staining, cells were incubated with blocking buffer (R37620, ThermoFisher) for 1 h at room temperature and then incubated with 1 µΜ GPC1 molecular beacons (MBs) for 1 h at 37 °C. After washing three times with PBS, cells were sequentially incubated with different colors of fluorescence‐labeled monoclonal antibodies (e.g., anti‐Rab7, anti‐GPC1, or anti‐ARF6) in 1% bovine serum albumin (BSA) solution after blocking with 5% BSA in PBS solution for 1 h at room temperature. After washing, cell fluorescence images were taken using TIRF microscopy. Cell co‐localization was further quantitated using the Imaris software (BITPLANE, Oxford Instruments, Zurich, Switzerland). First, a co‐localized area σ of Ch1 (e.g., anti‐Rab7 as ILV/MVB marker or ARF6 as microvesicle marker) and Ch2 (e.g., GPC1 mRNA or GPC1 protein probes) were generated and then further calculated the proportion by pixel analysis as:
$$\%GPC1mRNAin\frac{ILV}{MVB}=\frac{Colocalizationarea}{EntireareafromCh1of\frac{ILV}{MVB}}$$

All cell counting (cells/mm²) was executed by two different, blind to the experiment investigators, on six areas per section (which contained GFP⁺ cells) spaced at least 50 μm apart. Only Dapi⁺ and BrdU⁺ nuclei were taken into account. Images were captured using an Axioplan‐2 optical/widefield fluorescent (Zeiss) while confocal images were captured with Nikon Eclipse Ti and were assembled in ImageJ (Fiji) software. Demyelination, axonal loss, and inflammatory cell evaluation were analytically described in our previous work [27 (link)]. Connexin immunoreactivity was quantified with Bitplane Imaris software (Oxford Instruments) from analysis of multidimensional microscopy datasets. 3D reconstruction and visualization of the cells and connexins were rendered through the program from multiple z stacks. GJ plaques (Cx32 and Cx47) were defined as a concentration of connexin signal with size limits between 0.1 and 1 μm² [28 (link)]. The total number of connexin GJ plaques (Cx32 and Cx47) was measured in each image and then a ratio to the respective cell (BrdU⁺) was calculated, as previously described [29 (link)].

The illustration of behaviors in Fig. 1 and model in Fig. S9b were created with BioRender (BioRender.com). Quantification of 50 to 60 min LTP magnitude compared with the 10 min baseline, Data: mean ± SEM. Statistical significance was performed with two-tailed Student’s t test, ANOVA, or mixed effects model (restricted maximum likelihood). Dunnet’s, Tukey’s or Sidak’s multiple comparisons tests were used following significance in ANOVA or mixed effects model tests. Several outlier data points were excluded in behavior data, using outlier criteria of > 3 standard deviations outside of group mean. *p < 0.05, **p < 0.01, ***p < 0.001.The imaged figures were analyzed by ZEN (blue version) from Zeiss and Image J (Fiji) from NIH. Statistical significances were assessed using Student’s t test using GraphPad or R, the figures were draw by Graphpad or R, too. The bedgraph files in sequencing were examined by IGV. Tracing of dendrites and spines of GFP positive adult-born neurons was performed using Bitplane Imaris software (Oxford instruments) and Image J (FIJI distribution, NIH) with SNT plugin. The traced neurons were compared and draw by Inkscape (https://inkscape.org/).

Primary culture of E16.5 hippocampal neurons from non‐Tg and Mpst‐Tg mice were performed as previously described (Morikawa et al, 2018). Briefly, dissociated hippocampal neurons were plated on chambered coverslips coated with polyethyleneimine at 1 × 10⁵ cells per well and cultured in a 5% CO₂ atmosphere at 37°C. Cultured hippocampal neurons at DIV 14–21 were transfected with an EGFP expression vector using the calcium phosphate method for 2 days before the observation. The dendritic spines were observed using a confocal laser‐scanning microscope (LSM780) equipped with an Airyscan module (ZEISS). Data analyses were performed using IMARIS (BITPLANE) software (Oxford Instruments, Abingdon, UK).