90i upright widefield microscope

Imaging was performed with an SP5 inverted confocal microscope (Leica), a Nikon 90i upright wide-field microscope, or a Spinning-disk confocal microscope with constant parameters. For BrdU analysis, one 40 µm coronal section every 200 µm was quantified. BrdU counting in the granule cell layer (GCL) and glomerular layer (GL) from the same mice was performed on six sections per animal from bregma +4.0 to its anterior end using an in-house ImageJ macro that automatically detects cells within an ROI. The cell number was normalized by the ROI area for each image; the mean for each animal was reported. This method was validated in initial experiments where counting by hand in parallel gave similar results. BrdU counting in the V-SVZ was performed by hand on 8 × 40 µm coronal sections every 200 µm per animal, from bregma +1.34 to bregma −0.58mm, which represents the anterior V-SVZ in which proliferation is more sustained (Fiorelli et al., 2015 (link)). Cell numbers were normalized by ventricular wall length.

Monolayers were grown in Ibidi μdishes (Thistle Scientific) or Lab-Tek II chamber slides (Thermo) and allowed to proliferate for 24 h. Cells were treated as stated in the main text, washed with PBS and fixed in paraformaldehyde (4%, 10 mins). For samples co-stained with DAPI (500 nM, 2 min) this was added after fixation and membrane-permeabilisation (0.1% Triton, 10 mins) steps. Epi-fluorescent images were obtained using a Nikon 90i upright widefield microscope and a x60 oil-immersion objective. An in-house filter cube comprising FITC excitation with TRITC emission was employed to detect MLCT emission of 1 and 2. Confocal images were obtained using a Zeiss LSM 780 META inverted confocal microscope and x40 or x63 oil-immersion objectives. Microscopy images were processed using either Zeiss LSM Image Browser or ImageJ software.

Inflated lungs (30 cm water pressure) were fixed in 10% formalin immediately following removal. After a series of alcohol and xylene washes, tissues were blocked in paraffin. Paraffin sections were stained with hematoxylin & eosin (H&E) and imaged with a Nikon 90i Upright Widefield Microscope (Nikon Instruments Inc., Melville, NY). 100 µm scale bar added using Nikon Elements Software.