Sodium azide

Immunostaining of rat brains implanted with MEAs was conducted as previously described [21 (link)]. In brief, rats were euthanized 16 weeks post-implantation via intraperitoneal injection of sodium pentobarbital (Virbac Corporation, Westlake, TX, USA) and transcardially perfused with 1× PBS followed by 4% paraformaldehyde (PFA) (Sigma-Aldrich, St. Louis, MO, USA). The skulls were then submerged in 4% PFA for 48 h prior to extraction of the MEAs. Brains were extracted and stored in PBS and sodium azide for 24 h before initiating the histological process. Brain tissue was embedded in agarose gel, sliced into 100 µm sections, and stored in PBS and sodium azide at 4 °C overnight. The immunohistochemistry steps were as follows:
Day 1: Slices were treated with sodium borohydride (Sigma-Aldrich, MO, USA) to quench autofluorescence; blocked and permeabilized in a solution containing 4% normal goat serum (Abcam Inc., Waltham, MA, USA), 0.3% Triton X-100 (Sigma-Aldrich, MO, USA), 0.1% sodium azide (Sigma-Aldrich, MO, USA) in PBS, and 2% bovine serum albumin (Sigma-Aldrich, MO, USA); then, they were treated with Image-iT (Thermo Fisher Scientific, Waltham, MA, USA) followed by primary antibody treatment and stored at 4 °C overnight.
Day 2: Slices were washed with PBS and Triton X-100 and incubated in the secondary antibody solution for 2 h (Abcam Inc., MA, USA). Tissue samples were then mounted on glass slides and covered with coverslips. The antibodies used are summarized in Table 2.

Cisplatin staining and fixation of cells for CyTOF analysis was performed as previously described.^{18 (link),27 (link),41 (link),102 (link),103 (link)} Briefly, up to 6 million cells were resuspended in 2 mL contaminant-free PBS (Rockland) with 2 mM EDTA (Corning). Cells were then incubated for 1 min with an additional 2 mL of PBS/EDTA supplemented with 12.5 μM Cisplatin (Sigma-Aldrich). The Cisplatin staining was then immediately quenched with 10 mL CyFACS (contaminant-free PBS [Rockland] supplemented with 0.1% bovine serum albumin [BSA; Sigma-Aldrich] and 0.1% sodium azide [Sigma-Aldrich]). Cells were then fixed for 10 min at room temperature in 2% PFA (Electron Microscopy Sciences) diluted in contaminant-free PBS. After washing the cells in CyFACS, cells were resuspended in 100 µL of CyFACS containing 10% DMSO, and stored at –80°C until analysis by CyTOF.

Histological experiments were conducted to: (1) examine viral infection in the MPOA of mice used in behavioral experiments (Slc32a1^Flp::Esr1^lox/lox or Slc17a6^Flp::Esr1^lox/lox), (2) validate the specificity of AAV-frtFlex-Cre in Esr1^lox/lox mice, and (3) confirm the specificity of reporter gene expression in Slc32a1^Flp::Esr1-Cre::RC-FLTG mice. Mice were deeply anesthetized with pentobarbital and transcardially perfused with 40 mL of 4% paraformaldehyde (PFA) in PBS. Brains were extracted and post-fixed in 4% PFA in PBS on ice for 5–7 hours before being transferred to PBS with 0.05% sodium azide (Sigma) for storage at 4°C until sectioning. Free-floating coronal brain sections (50 μm thick) were prepared using a vibratome (Leica) and stored in PBS with 0.05% sodium azide until immunohistochemistry (IHC). Sections were first washed in PBS (3 × 5 minutes) and blocked with 15% normal donkey serum (NDS) in PBST (0.3% Triton X-100) for 2 hours at room temperature. Following blocking, sections were incubated with an anti-Esr1 primary antibody (1:500, Santa Cruz, sc-542) diluted in 15% NDS in PBST for 72 hours at 4°C. After incubation, sections were washed in PBST (0.3% Triton X-100, 3 × 30 minutes) and incubated with a secondary antibody (1:500, Life Technologies donkey anti-rabbit 568 or Jackson ImmunoResearch donkey anti-rabbit 647) diluted in 15% NDS in PBST for 2 hours at room temperature. Sections were then washed in PBST (2 × 15 minutes), incubated with DAPI for 2 minutes, rinsed in PBS (2 × 15 minutes), mounted on slides, and coverslipped with mounting medium. Tiled images of the MPOA were acquired using a Zeiss ApoTome2 fluorescence microscope with a 20x objective and Zen software (Zeiss). Image acquisition settings were consistent across all experiments. The resulting CZI files were analyzed with HALO software using the ISH-IF version 1.2 module (Indica Labs). Regions of interest (ROIs) corresponding to the MPOA were manually outlined in both hemispheres across three adjacent sections from each brain. GFP- or YFP-positive cells, Esr1-positive cells, and double-positive cells were automatically detected and quantified using specific threshold settings to define phenotypes.

Gravid fmo-4p::mCherry transcriptional reporter adult animals were placed on NGM plates seeded with E. coli OP50. After 3 hr, the adults were removed, and the eggs were allowed to develop at 20 °C until they reached late L4/young adult stage. Then 30 worms were transferred to NGM plates seeded with E. coli OP50 topically spotted with 150 µL of 0.5 M ethylenediaminetetraacetic acid (EDTA) (ThermoFisher, AM9260G) or 150 µL of water. An optimal concentration of EDTA was determined by first assessing survivability of worms exposed to a range of concentrations and then by assessing the fluorescence of the fmo-4p::mCherry reporter worms exposed to a range of concentrations. The worms were incubated for 24 hr at 20 °C. After 24 hr, ~20 worms per condition were then picked off these plates and added to unseeded NGM plates, anesthetized in 0.5 M sodium azide (Sigma), and imaged at 6.3 x magnification with the LASx software and Leica scope using the mCherry fluorescence channel (Miller et al., 2022 (link)). Three replicates were performed. Each worm was recorded in ImageJ. Data were analyzed in GraphPad Prism using t tests.

At the stage of 1 day of adulthood, synchronized gst-4::GFP transgenic worms fed heat-killed E. coli OP50 and supplemented with wheat extracts from embryo hatching were anesthetized with sodium azide (20 mmol L⁻¹) (Sigma-Aldrich, St. Louis, MO, USA) and observed by Zeiss Axiovert 25 microscope (Zeiss, Oberkochen, Germany) as described in Bianchi et al., 2020 [11 (link)]. The experiments were repeated three times and 15 worms per group were used in each experiment. Images were taken at the time of exposure of 0.2 s and fluorescence was analyzed using ImageJ software 1.54k. Scale bars were inserted by Zeiss ZEN Microscopy Software 2011.