Ar1 confocal microscope

Microscopy image acquisition was performed using an Olympus BX-51 microscope (Olympus corporation, Tokyo, Japan) or multi-photon Nikon AR1 confocal microscope. Chromagen labelling of AR receptors was imaged with brightfield light microscopy using a 10× objective, and ImageJ software (National Institutes of Health, Bethesda, Maryland, USA) was used to assist in the quantification of the number of AR-positive nuclei in two representative sections from each nucleus analysed (AVPV, PeN, rARN, mARN and cARN).
Following double immunofluorescent labelling of vGAT and GFP in GnRH-GFP VEH control and PNAM mice, 10 GnRH neurons were selected at random from two representative sections of the rPOA. GnRH neurons were imaged using a Nikon A1R multi-photon confocal microscope with 488 (HV: 80, Offset: 0, Laser: 1.5) and 543 nm (HV: 85, Offset: −5, Laser: 2.0) diode lasers. Sections were imaged using a Plan NeoFluor 40× oil objective, taking Z-stack images every 0.6 µm (pinhole at 1.1 AU) with a 2× zoom function (Nikon® Instruments Inc., Tokyo, Japan). As previously described, vGAT apposition and GnRH spine density was analysed at the GnRH neuron soma and in 15 μm intervals of the primary dendrite out to 75 μm^{23 (link),24 (link)}.

Undifferentiated colonies of hPSCs as well as differentiated retinal organoids derived from all cell lines were fixed using 4% paraformaldehyde in PBS for 30 min, and then washed three times with 1X PBS. For retinal organoids, samples were then equilibrated in 20% sucrose followed by 30% sucrose solutions. They were then embedded in OCT and flash frozen followed by cryosectioning at 12 μM thickness. Immunocytochemistry for all samples was then performed by permeabilizing samples using 0.2% Triton X-100 for 10 min followed by 3 washes with 1X PBS. Samples were then blocked using 10% donkey serum for 1 h at room temperature, followed by incubation with the respective primary antibodies diluted in 5% donkey serum and 0.1% Triton X-100 overnight at 4 °C. Primary antibodies used are listed in Supplemental Table 1. The next day, samples were washed 3 × in PBS before incubation with corresponding secondary antibodies in 5% donkey serum and 0.1% Triton X-100 for 1 h at room temperature. Following 3 more washes in PBS, samples were mounted on slides for visualization either on a Leica DM5500 upright microscope or a Nikon AR1 confocal microscope.