Leica application suite x imaging software

To visualize microglia and neurons, sections (Bregma - 1.5 mm) were incubated with rabbit anti-Iba1 (1:1000; Wako) or rabbit anti-NeuN (1:1000; Millipore) primary antibody, respectively, followed by Alexa Fluor 647 donkey anti-rabbit IgG secondary antibody (1:500, Invitrogen). In order to visualize microglial lysosomes, sections were incubated with rabbit anti-Iba1 (1:1000; Wako) and rat anti-CD68 (1:500; Abcam) primary antibodies, Alexa Fluor 594 donkey anti-rabbit IgG and Alexa Fluor 647 donkey anti-rat IgG secondary antibodies (1:500, Invitrogen), and 0.1% Sudan Black B (Sigma-Aldrich) solution in 70% ethanol for 2 min prior to cover-slipping. Slides were then imaged using a Leica SP8 upright confocal microscope at 63X magnification and sequential optical sections captured using the Leica Application Suite X imaging software. Lipofuscin was imaged at 488 nm excitation and 495–545 nm emission. Sequential optical sections were analyzed using ImageJ software (NIH). For each animal, 2–4 images were quantified and averaged, and these values were used to calculate the mean and standard error for each experimental group.

Confocal laser scanning microscopy (CLSM) analysis was performed to determine the cell density and morphology on the surface of the material. The hBMSCs seeded on FU scaffolds (n = 3) and cBS (n = 3) on Day 14 were stained using Alexa Fluor 488 phallotoxins F-actin and counterstained with Hoechst 33342 nucleic acid dye (Life Technologies, Invitrogen). FU scaffold and cBS without cells was used as a control. Samples were stained according to the protocol provided by manufacturer. After 20 min of incubation, the samples were washed with PBS (1X). All images were acquired using Leica TCS SP8 X inverted confocal microscope with X20/0.40 NA objective lens and 2X-zoom (Leica) and analysed using Leica Application Suite X imaging software (Leica LAS X, UK).

Colocalization analysis of GFP and bipolar cell subtype markers was performed using Leica Application Suite X imaging software (version 3.5.5., Leica). Cells were counted in 100 μm × 100 μm regions of interest. Counting of cells in the region of interest was performed independently by two to four people under the condition that the origin of tissue, region, and eccentricity were blinded. We used a manual cell counter plugin included with the Fiji distribution of ImageJ (Schindelin et al., 2012 (link)). We counted GFP-expressing bipolar cells only if the somas resided in the outer half of the INL. For antibody-stained cells, bipolar cells were determined and counted if their somas resided in the outer half of the INL and dendrites extending to the outer plexiform layer (OPL). All statistical analysis and graph preparation were performed using GraphPad Prism (version 9, GraphPad Software, La Jolla, CA, United States). Two-way ANOVA was used to analyze the bipolar cell topography data for orientation and eccentricity with Tukey’s multiple comparisons test. Linear regression was used to evaluate density gradients along the ventral-dorsal and nasal-temporal axes. Differences were considered significant if p < 0.05. SigmaPlot (version 14, Systat Software, San Jose, CA) was used to make color-coded heat maps using coordinates of counted fields and calculated densities.