Vibratome

Embryos with wounded corneas were collected at 3, 5, 8, 9, 10 and 11 days post wounding (dpw). Following decapitation, eyes were collected in Ringer’s solution and fixed overnight in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), pH 7.2. Corneas were dissected from the surrounding scleral tissue, mounted on glass coverslips with 50% glycerol in PBS (v/v) and imaged en face for whole-mount imaging. For cross-section imaging, corneas were embedded in 10% low melting point agarose (NuSieve GTG; Lonza, Rockland, ME, USA) as previously described^{52 (link)}. A schematic representation of the wounded embryonic cornea, the tissue orientation, and imaging approaches is illustrated in Fig. 1. Tissue sections of approximately 300 µm thick were cut using a vibratome (Campden Instruments Ltd.) and imaged. The wounded corneas were grouped according to the different phases of the wound healing process; early (3–5 dpw), mid (8–9 dpw) and late (10–11 dpw) healing. At least three wounded and unwounded (control) corneas were analyzed in each group.

Npy-hrGFP mice were euthanized by decapitation, and the brains rapidly removed and sliced on a vibratome (Campden Instruments, Loughborough, UK) in ice-cold oxygenated incubation artificial cerebrospinal fluid (aCSF) containing (mM): 95 NaCl, 1.8 KCl, 1.2 KH₂PO₄, 7 MgSO₄, 26 NaHCO₃, 0.5 CaCl₂, 15 glucose, and 50 sucrose. Slices were perfused with room temperature recording aCSF containing (mM): 127 NaCl, 1.8 KCl, 1.2 KH₂PO₄, 1.3 MgSO₄, 26 NaHCO₃, 2.4 CaCl₂, and 5 glucose. Npy-hrGFP neurons were visualized in 250-µm slices using an Olympus BX51 with inbuilt infrared video-enhanced differential interference contrast optics and GFP fluorescence optics. Neurons were patched using 7 to 10 MΩ pipettes containing (mM): 130 K-gluconate, 10 KCl, 2 MgCl_2, 10 HEPES, 0.5 EGTA, 2 K₂ATP, and 0.5 NaGTP. Data were recorded on an Axoclamp 2A amplifier in bridge mode (Molecular Devices, San Jose, CA) and a CED 1401 A/D data acquisition interface (CED, Cambridge, UK) to give current clamp data at 30 kHz sampling frequency. NNC0165-1273 (Novo Nordisk, Maaloev, Denmark) was diluted in aCSF to a concentration of 50 nM and applied to perfusion chamber through a gravity-driven system. Effects were sampled with no current manipulations. All applications were timed for 3 to 5 minutes and perfusion flow was maintained at standard 1 to 2 mL/min⁻¹.

All quantitative assessments of BRM cell or PC distribution, including calculations of clustering L values and cell densities in the alveoli or in uninfected and infected sites, were done by analyzing large tiles of static lung sections using two-photon excitation laser scanning microscopy (TPLSM), as previously described (MacLean et al., 2022 (link)). Briefly, after euthanization with pentobarbital, mouse lungs were inflated with 1 ml of 30–35°C low-melt agarose (1% in PBS) into the lungs. Individual lobes were dissected and stored in PBS before sectioning at 450 mm on a vibratome (Campden Instruments Ltd.). Sections were mounted on plastic coverslips using Vetbond tissue glue (3M), placed on ice in PBS, and immediately imaged. Sections were imaged on a Zeiss LSM880 microscope, using a primary excitation wavelength of 930 nm, with an Apochromat 20 × 1.0 DIC VIS-IR D = 0/0 (UV) objective lens.

At the end of the microdialysis experiment, mice were deeply anesthetized and sacrificed. The probes were removed and the brains stored in formalin (8%) for histological examination to verify the correct placement of the microdialysis probe. Brains were cut with a vibratome (Campden Instruments, Leics, UK) in serial coronal slices oriented according to the mouse brain atlas of Paxinos & Watson (1998), and the location of the probes was reconstructed.

Hippocampal slices were prepared as previously described (Kim et al. 2012; Owen and Grover 2015). Male and female Sprague–Dawley rats (30–60 days old, Hilltop Lab Animals, Scottdale, PA) were sedated by CO₂/air inhalation, and decapitated. The brain was removed and placed into chilled artificial cerebrospinal fluid (ACSF) composed of (in mmol/L): 124 NaCl, 26 NaHCO₃, 3.4 KCl, 1.2 NaH₂PO₄, 2.0 CaCl₂, 2.0 MgSO₄, 10 glucose (pH 7.35, equilibrated with 95% O₂/5% CO₂). A block containing both hippocampi was glued to the stage of a vibratome (Campden Instruments, Lafayette, IN), immersed in chilled ACSF, and sectioned into 400–500 μm thick slices in the coronal or horizontal plane. Slices were dissected to remove the hippocampus from surrounding structures. Hippocampal slices were stored at room temperature (20–22°C) in an interface holding chamber. For recordings, individual slices were transferred to a small volume (approximately 200 μL) interface recording chamber heated to 34.5–35.5°C. Slices were perfused with oxygenated ACSF at a rate of 1–1.5 mL/min. All procedures were approved by the Institutional Animal Care and Use Committee at Marshall University.