Zen black 2 3 sp1

Super-resolution imaging was performed in the Neuroscience Light Imaging Facility (NINDS) with a confocal laser scanning microscope Zeiss LSM 880 (Carl Zeiss AG, Oberkochen, Germany) equipped with a 32 channel Airyscan detector (Korobchevskaya et al., 2017 (link)). The whole organ of Corti images were taken in SR mode with a 20X objective (Carl Zeiss). To image the hair cells, we collected a z-stack of images from the stereocilia to the apical half of the hair cell body. We used oil immersion alpha Plan-Apochromat 63X/1.4 Oil Corr M27 objective (Carl Zeiss) and Immersol 518F immersion media (n_e = 1.518 (30°), Carl Zeiss). Identical image acquisition settings, no averaging, and optimal parameters for x, y, and z resolution were used in all samples from each independent experiment. Image acquisition and Airyscan image processing were done with Zen Black 2.3 SP1 software (Carl Zeiss) using the Airyscan 3D reconstruction algorithm with the automatic default Wiener filter settings.
Confocal imaging on transfected HEK293 cells was performed in the Microscopy and Imaging Core (NICHD) with an inverted laser scanning microscope Zeiss LSM 780 (Carl Zeiss) equipped with a motorized stage, definite focus and a high sensitivity GaAsp multi-channel spectral detector. We used a 63X/1.4 objective Plan-Apochromat (Carl Zeiss) and the Zen software (Carl Zeiss) for image acquisition.

Confocal images were acquired with a Zeiss LSM 700, using a EC Plan-Neofluar 40× oil (N.A.1.30) or Plan-Apochromat 63× oil (N.A. 1.40) objective and 488-, 555-, and 633-nm laser excitation lines. Images were acquired as z-stacks containing planes at 0.5-μm interval, with 0.1-μm pixel size and 2× pixel averaging. All images are displayed as maximum intensity projections. Images in Extended Data Figure 1-2 were acquired as tile scans, and stitched using Zeiss Zen Black 2.3 SP1 software.

3 x 10⁴ HEK293T cells transiently transfected with CXCR3-A, CXCR3-B, ACKR3, or ACKR2 C-terminally fused to mNeonGreen were seeded in a poly-lysine-coated µ-Slide 8-well-chambered coverslip (Ibidi). After 24 hours, cells were washed twice with PBS and fixed with 3.5% (w/v) paraformaldehyde solution for 20 minutes at RT. Cells were washed three times with PBS and incubated with anti-CXCR3 (1C6), anti-ACKR3 (8F11-M16), and anti-ACKR2 (196124) mAb for one hour at 4°C. Cells were then washed twice with PBS and incubated for 20 minutes at RT with Hoechst 33342 dye (1 µg/mL). Cells were washed twice with PBS before acquiring images on a Zeiss LSM880 confocal microscope using a 63x oil-immersion objective and Zen Black 2.3 SP1 software (Zeiss).