Vibratome

Fresh lung specimens were obtained from four IPF patients that underwent lung transplantation. Precision-cut lung slices were prepared in two ways: lung specimens were cut into strips (length: 2–3 cm, thickness: 3–5 mm) and later chopped into 200-µm-thick slices using a McIlwain Tissue Chopper (Campden Instruments Ltd.); or lung tissues were gently injected with 1.5% low-melting agarose (Roth) and cut using a vibratome (Thermo Fisher Scientific) into 400-µm-thick slices. Five to six PCLS were cultured in 5 mL of DMEM supplemented with 10% BCS at 37 °C and 5% CO₂ for 5 days. Cultures were treated with different agents at the beginning of the culture process.

For electrophysiology, mice were sacrificed during the light phase at ZT4 and during the dark phase at ZT16. In order to perform all the electrophysiological recordings during the working time of the day, mice sacrificed at ZT16 received a light/dark cycle inversion (light on at 19:00) for at least 14 days prior to start the control chow or PLX5622 treatment, for microglia depletion in wild‐type mice. In this way, slices were harvested at 11:00 for all groups. To verify that light/dark cycle inversion did not affect the results of the experiments, a group of control mice receiving the normal light/dark cycle was sacrificed at 23:00. No significant differences were observed between the ZT16 groups in normal and inverted cycles and data were pooled.
For slicing preparation, anesthetized animals were decapitated and the whole brains were rapidly removed from the skull and immersed for 10 min in ice‐cold artificial cerebrospinal fluid (ACSF; composition in mM: NaCl 125, KCl 4, CaCl₂ 2.5, MgSO₄ 1.5, NaH₂PO₄ 1, NaHCO₃ 26, and glucose 10; 295–300 mOsm), continuously oxygenated with 95% O₂ and 5% CO₂ to maintain the proper pH (7.4). Transverse 350 μm slices were cut at 4°C with a vibratome (Thermo Scientific, USA) and then placed in a chamber containing oxygenated ACSF. After their preparation, slices were allowed to recover for at least 1 h at 30°C.

After recordings the animals were deeply anesthetized with urethane (3 g/kg, intraperitoneally) and perfused intracardially with 4% paraformaldehyde and 1% glutaraldehyde (Sigma). The brains were removed and left for fixation for a few days. One-hundred micron-thick coronal slices were cut using a Vibratome (Thermo Fisher Scientific, Waltham, MA, USA). Electrode positions were identified from the DiI tracks overlaid on the microphotographs of sections after cresyl violet staining.

Animals were anesthetized with halothane and then decapitated. The whole brain was rapidly removed and immersed in ice-cold artificial cerebrospinal fluid (ACSF) solution containing (in mM): NaCl 125, KCl 4.4, CaCl₂ 2.5, MgSO₄ 1.5, NaHPO₄ 1, NaHCO₃ 26 and glucose 10 which was oxygenated with 95% O₂, 5% CO₂ to maintain a pH close to 7.4. Hippocampi were rapidly removed from the brain and elongated (Fig. 1a). The dorsal (or septal) hippocampus was identified by its proximity to the retrosplenial cortex and the ventral (or temporal) was close to the amygdaloid complex. Isolated hippocampi were placed in an Agar block (2.25% weight/volume in bi-distilled water) and fixed on a plate (Fig. 1b) for slicing with a vibratome (Thermo Scientific, USA). Transverse 350 μm thick slices were cut at 4 °C from the entire length of each hippocampus except the extremities of length about 250 μm. The longitudinal position of each slice was identified as distance, in μm, from the dorsal pole before recording. Results are presented with septo-temporal recording sites schematically divided into DH, IH and VH regions of 2 mm each (Fig. 1c). After preparation, slices recovered for 1 h at 30 °C in a chamber containing oxygenated ACSF. Importantly, the slice isolation procedure seems not preclude functional differentiation along the septo-temporal axis.

Luminescence was measured from cultured ARC/ME slices of heterozygous PER2::LUC mice as described previously (Yoo et al., 2004 (link)) and Guilding et al., 2009 (link)). Briefly, brains were isolated and harvested in ice-cold Hank’s balanced salt solution (HBSS). 300 µm ARC/ME containing coronal slices (−1.80 mm and −2.10 mm relative to bregma) were prepared using a vibratome (Thermo Scientific, MA, USA). Bilateral ARC/ME regions were further dissected under a dissecting microscope and then immediately placed onto a culture plate insert (Merck Millipore) in 35 mm Petri dishes filled with 1 ml recording medium (same as the one used for cell culture). Luminescence was measured in a LumiCycle (Actimetrics) at 32.5°C.

For intraocular injections, mice were anesthetized with isoflurane or hypothermia, and 1–2 μl of 1 mg/ml CTB was injected into the eye intravitreally with a fine glass pipette attached to a picospritzer. After 2 days, perfused and PFA fixed brains were sectioned (90 μm) using a Vibratome (HM650v, ThermoFisher). Sections were stained with DAPI and mounted with Vectashield (Vector Laboratories, Burlingame, CA). Images were acquired on a Zeiss LSM 700 confocal microscope. A similar approach was used to inject 1–2 μl of a 1:1 mixture of the following AAVs into the eyes: AAV9.hEF1a.lox.TagBFP.lox.eYFP.lox.WPRE.hGH-InvBYF (AV-9-PV2453, 3.47e13 gc/ml) and AAV9.hEF1a.lox.mCherry.lox.mTFP1.lox.WPRE.hGH-InvCheTF (AV-9-PV2454, 1.04e13 gc/ml). AAVs were injected into the eyes of P0 or P12 mice and 1–2 weeks after the injection, mice were anesthetized, perfused, and their brains were fixed in 4% PFA overnight. Brains were then sectioned (90 μm) using a Vibratome and sections were mounted with Vectashield. Images were acquired on a Zeiss LSM 700 confocal microscope.