Metafer vslide scanner

Mice were administed an overdose of sodium pentobarbitone and transcardially perfused with 40 ml of phosphate-buffered saline (PBS), followed by 45 ml of 4% paraformaldehyde in PBS for fixation. The brain was extracted and fixed at 4 °C for 12–24 h. It was then washed once with PBS and transferred to a 30% sucrose solution for 36 h prior to sectioning. 40 μm thick sections were cut using a sliding microtome and collected in a 1:6 series. Cell nuclei were stained by 4’,6-diamidino-2-phenylindole (DAPI, catalog #6329; Sigma Aldrich). Sections were first washed once in PBS for 10 min, and then incubated in 1:5000 DAPI-PBS solution for 15 min at room temperature. After two washes, the sections were mounted on SuperFrost slides using fluorescence mounting medium (Dako, Agilent). Images were captured using a slide scanner (Metafer VSlide Scanner, MetaSystems) and microscope (Axio Imager Z2, Zeiss) with a 20 × 0.8 NA/0.55 mm objective lens.

To visualize p75NTR⁺ cells in the basal forebrain nuclei, images were acquired using an upright fluorescence slide scanner (Metafer VSlide Scanner by MetaSystems using Zeiss Axio Imager Z2) with a 20× air objective. p75NTR⁺ cells in the MS/DBB, NBM and SI were quantified using Imaris software (Version 9.3). For experiments evaluating neurogenesis and adult-born neuron morphology, only animals with more than a 50% loss of p75NTR⁺ cells in the MS/DBB following p75-Sap injection were included in the study. Fluorescence immunolabeling in tissue was visualized and imaged using a Nikon Plan Apochromat 20×/0.75 NA air objective and a Plan Apo Lambda 60x/1.4 NA oil-immersion objective on a Diskovery spinning disk confocal microscope. Representative images of BrdU, DCX, Nestin-GFP, Iba1 or tdTomato staining in the dentate gyrus were taken using a 20× objective at 0.5-2 μm intervals. For morphological analysis, three-dimensional images of dendrites were obtained from z-stacks of confocal images taken at 0.5 μm intervals using a 60× oil immersion objective. To accurately reconstruct the dendritic morphology, we selected tdTom⁺ newborn neurons that were entirely contained within the tissue section. Morphological analysis was performed using the Imaris software.

Imaging was performed on an Axio Imager upright fluorescence microscope (Zeiss) and Metafer VSlide Scanner (Metasystems) using Zeiss Axio Imager Z2. All measurements and analyses were performed using Imaris 7.2.3 software (Bitplane).