Inverted axio observer z 1

Brain tissue was processed according to standard procedures. In brief, two to three weeks after AAV injection (for anterograde tracing) or one week after RV injection (for retrograde tracing), mice were deeply anesthetized with isoflurane and immediately perfused intracardially with ~15 ml of phosphate-buffered saline (PBS) (pH 7.2) followed by ~15 ml of 4% paraformaldehyde (PFA) in PBS. Brain tissue was carefully removed, post-fixed in 4% PFA in PBS at 4°C overnight, dehydrated in 30% sucrose in PBS for 48 hr, and embedded in Tissue Freezing Medium (Triangle Biomedical Sciences, Cincinnati, OH). Brains were cut in 30 or 50 µm coronal sections using a cryostat (Thermo Scientific, Waltham, MA) and mounted with VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA) or DAPI Fluoromount-G (Southern Biotech, Birmingham, AL). One out of every three sections were imaged using 20X/0.75 objective in a high-throughput slide scanner (Nanozoomer-2.0RS, Hamamatsu, Japan) for further processing. We also imaged selected brain regions (Figures 2 and 3) using a Zeiss (Germany) inverted AxioObserver Z1 fully motorized microscope with LSM 710 confocal scanhead, 10X/0.3 EC Plan Neofluar M277 objective or a 20X/0.8 Plan Apochromat M27 objective.

ER was visualized by staining with the ER-specific dye, ER Tracker™ Blue/White DPX (Thermo Fisher Scientific, Cat. No E12353), which is retained within the ER lumen, thus labeling the ER tubular network, according to the manufacturer’s instructions. Briefly, cells were seeded on glass coverslips and cultured for 24 h and then incubated for 30 min at 37°C and 5% CO₂ with 1 μM ER tracker diluted in the culture medium. Then, the stained cells were fixed with 4% formaldehyde for 10 min, washed in PBS, and mounted using VECTASHIELD PLUS Antifade Mounting Medium without DAPI. Images were collected with the Zeiss LSM780, inverted Axio Observer Z.1 equipped with the 63x/1.4 Oil Plan-Apochromat DIC objective. A diode laser of 405 nm was used to excite fluorescence. Optical sections (2048 pixels × 2048 pixels × 8 Bit/pixel) were collected. The images were processed using ZEN Blue 2.1 software.

Microscopically, EGFP⁺ cells were identified by using an inverted fluorescence microscope (Zeiss Axiovert 25) or a confocal laser scanning microscope (Zeiss inverted AxioObserver Z1 equipped with LSM 700 scanning module). Nasal septum used for indirect immunofluorescence staining was transferred to PBS, permeabilized with 0.1% (v/v) Triton-X100 for 30 min and subsequently stained. TO-PRO-3 (Invitrogen) was used to counterstain nuclei (red). All images were generated using Zen 2010 software (Zeiss).