Zen black imaging software

The number of neurons in level-matched L4 DRG of E12 Cd40^−/− and Cd40^+/+ embryos was quantified using a combined stereological and confocal approach (Howell et al., 2002 (link); Yoshikawa et al., 2007 (link)). The DRG were fixed in 4% PFA and cryoprotected in 30% sucrose before being serially sectioned at 10 μm. Sections were blocked in 5% BSA (Sigma-Aldrich) for 1 h and were then incubated overnight at 4°C with rabbit monoclonal anti-NeuN (1:500, ab177487, Abcam) and mouse monoclonal anti-βIII-tubulin (1:500, MAB1195, R&D Systems) in PBS containing 1% BSA. After gentle washing with PBS, the sections were incubated with fluorophore-conjugated secondary antibody (1:500, Alexa Fluor 488/546, Thermo Fisher Scientific) in 1% BSA for 1 h in the dark at room temperature. Imaging was performed using a Zeiss LSM710 confocal microscope and the number of immunoreactive neurons were counted from the level-matched lumbar DRGs of each genotype. Using the 10 μm serial sections containing whole L4 DRGs at E12, the areas of DRG in each section were measured and the DRG volume was calculated using Zen Black imaging software (Zeiss).

Zebrafish embryos were decorionated using 1 mg/ml Protease from Streptomyces griseus (Sigma). For Lightsheet imaging (Zeiss), embryos were screened for fluorescence using a fluorescent microscope (Zeiss axio zoom V16), and mounted in a column of 1% low melting point agarose (Sigma). Zebrafish embryos were imaged using dual side illumination, with maximum intensity projection (MIP) processed images generated using the Zen Black imaging software (Zeiss).
For assessment of the relative proportions of red and green fluorescent cell populations, 10 embryos from each embryonic stage were imaged on a fluorescent microscope (Zeiss axio zoom V16) and pixel count for each fluorescent marker extracted using the ImageJ software package (27 (link)).

For life cell imaging, algae were synchronously grown under standard conditions, harvested on a 100-µm mesh, and transferred with a small amount of standard Volvox medium to an uncoated coverslip-bottom culture dish (Ibidi, Germany). A coverslip with four small dots of desiccator grease at the corners, which serve as a spacer, was cautiously placed on the specimen to slightly fix the algae, while preventing excessive compression. Specimens were freshly prepared and examined immediately using an inverted LSM780 confocal laser scanning microscope (Carl Zeiss GmbH, Germany) equipped with a LCI Plan Neofluar 63×/NA 1.3 objective (Carl Zeiss GmbH). A preheated incubation tool was used to keep the algae in the culture dish constantly at the standard temperature of 28°C. The exposure to laser light was kept as low as possible. Fluorescence of both YFP and chlorophyll was excited by an argon-ion (Ar⁺) laser at 514 nm. The emitted YFP fluorescence was detected at 517–553 nm, whereas chlorophyll fluorescence was detected at 651–700 nm. Fluorescence intensities were recorded for YFP and chlorophyll in two channels simultaneously. At the same time, transmission images were generated in a third channel using a transmission-photomultiplier tube (PMT) detector. Images were recorded with 12-bit depth using the ZEN black imaging software (ZEN 2011, Carl Zeiss GmbH, Germany).

Dark-field images for regional identification were obtained using Image-Pro Plus software (version 6.3) and an Olympus BX51 microscope equipped with a darkfield condenser at 12.5X (Supplemental Fig. S2). Borders of regions of interest were traced from darkfield images for later superimposition over fluorescent confocal images. Regions of interest included HVC outside of infarcts, RA, and Area X. Sections that contained portions of all three regions of interest were identified to ensure equal representation across treatment conditions. Fluorescent images were obtained using a Zeiss laser scanning microscope (LSM, 700 Axio Observer) with 40X (Plan-Apochromat/1.4 Oil DIC M27) and 10X objectives (EC Plan-Neofluar/0.30 M27). Using Zeiss ZEN Black imaging software, Z-stack images were compiled using 5 slices and analyzed after superimposing at maximum intensity using Image J.