The implantation of the hippocampal imaging window was carried out in mice aged 12–14 months as explained in previous reports4 (link),5 (link). Briefly, mice were deeply anesthetized with isoflurane (2% for induction and 1.5% for maintenance) and provided with analgesia (buprenorphine). The skin was opened, and the cranial bone was exposed and locally removed above the dorsal DG (− 2.0 mm posteriorly and −1.5 mm laterally from the bregma, 3 mm in diameter). The cortical tissue above the level of the corpus callosum (3 mm in diameter and 1.5 mm in depth) was then removed sequentially using a biopsy punch (Miltex) and a blunt 22-gauge needle for aspiration. The hippocampal imaging window (stainless steel cannula, 3 mm in diameter and 1.5 mm in height, covered by a glass coverslip; Warner Instruments) was inserted and stabilized in place using a stereotactic arm and stably fixed to the cranial bone with ultraviolet-cured dental cement (Ivoclar Vivadent) when bleeding stopped.
Glass coverslip
Manufactured by Warner Instruments
Sourced in United States
Glass coverslips are thin, transparent pieces of glass used to cover and protect samples in microscopy. They provide a flat, smooth surface for samples and allow for high-quality imaging and observation.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 568, by Thermo Fisher Scientific (3 mentions)
Triton x 100, by Merck Group (2 mentions)
Fluorsave, by Merck Group (2 mentions)
Nis elements software, by Nikon (2 mentions)
A1r mp multiphoton system, by Nikon (2 mentions)
D glucose, by Merck Group (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions)
Nahco3, by Merck Group (2 mentions)
Trypsin inhibitor, by Merck Group (2 mentions)
Withpoly l ornithine and laminin, by Merck Group (2 mentions)
Accutase, by Innovative Cell Technologies (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Solidworks, by Dassault Systèmes (1 mentions)
Fura 2 am, by Thermo Fisher Scientific (1 mentions)
Inverted fluorescence microscope, by Leica (1 mentions)
12 well cell culture plate, by Corning (1 mentions)
Metafluor software, by Universal Imaging (1 mentions)
Bovine calf serum (bcs), by Thomas Scientific (1 mentions)
17β estradiol, by Merck Group (1 mentions)
Matrigel, by Corning (1 mentions)
20 protocols using glass coverslip
1
Implantation of Hippocampal Imaging Window
2
Customizable Mammary Window Chamber Design
3
Biocytin-Filled Neuron Imaging Protocol
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Slices with biocytin-filled neurons were fixed in 4% PFA (in 0.1M PBS) overnight, then washed in PBS before incubating in PBS containing 2% Triton X-100 (Sigma) and streptavidin conjugated with Alexa568 (1:200, Invitrogen) for at least 14 hours at 4 °C. Slices were washed in PBS the next day and mounted onto a glass coverslip (#1.5, 22 × 40 mm, Warner Instruments), imaging side down. A well for the slice was crafted from two 1.5 mm thick glass coverslip (#1, 22 × 22 mm, Warner), which were stacked and attached to the short edges of the main coverslip. After removing excess PBS from the slice, drops of mounting media (FluorSave, Millipore) were applied before sealing with glass slide (75 × 25 × 1 mm, Ever Scientific). Once the mounting media solidified, the slide was sealed with nail polish.
Two-photon imaging was performed on a Nikon A1R MP+ Multiphoton system using a water-immersion 25× objective lens (APO LWD, N/A 1.10, WD 2.0 mm, optimized for coverglass thickness 0.17 mm, Nikon) with the laser (Ti:sapphire, Chameleon Vision, Coherent) tuned to 860 nm wavelength. Image stacks were acquired using Nikon NIS-Elements software, with settings of at 1024 × 1024 pixels (0.5 μm x-y pixel size), 2.4 μs dwell time, and 2 μm z-step size. The laser power and the gain of the GaAsP photomultiplier detectors were depth-adjusted.
Two-photon imaging was performed on a Nikon A1R MP+ Multiphoton system using a water-immersion 25× objective lens (APO LWD, N/A 1.10, WD 2.0 mm, optimized for coverglass thickness 0.17 mm, Nikon) with the laser (Ti:sapphire, Chameleon Vision, Coherent) tuned to 860 nm wavelength. Image stacks were acquired using Nikon NIS-Elements software, with settings of at 1024 × 1024 pixels (0.5 μm x-y pixel size), 2.4 μs dwell time, and 2 μm z-step size. The laser power and the gain of the GaAsP photomultiplier detectors were depth-adjusted.
4
Biocytin-Filled Neuron Imaging Protocol
5
Cytosolic Calcium Imaging with Fura-2/AM
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[Ca2+]cyt imaging experiments were performed as previously described (26 (link), 55 (link)). Cells were grown on glass coverslips (Warner Instruments) in 12-well cell culture plates (Corning Incorporated) for 24 h. [Ca2+]cyt was measured using Ca2+-sensitive fluorescent indicator fura-2/AM (Invitrogen). Cells were incubated with 5 μM fura-2/AM for 1 h in physiological salt solution (PSS) at 22 °C in the dark and then washed in PSS for 30 min. Thereafter, cells on coverslips were mounted in a standard perfusion chamber on the stage of an inverted fluorescence microscope (Leica). Fluorescence signals were imaged using an intensified CCD camera (ICCD200) attached to an inverted fluorescence microscope (Leica) and recorded with MetaFluor software (Universal Imaging Corporation). Images were acquired every 3 s. The dual wavelength excitation method for the measurement of fura-2 fluorescence was used. The excitation wavelengths were 340 and 380 nm, and the emitted fluorescence was collected at 510 nm. [Ca2+]cyt was presented as fluorescence ratios (F340/F380) after background subtraction. The PSS used in digital measurement contained the following: 140 mM Na+, 5 mM K+, 2 mM Ca2+, 147 mM Cl-, 10 mM Hepes, and 10 mM glucose (pH 7.4). The osmolality for the solution was ∼300 mosmol.kg−1 of H2O.
6
Murine Adipocyte Differentiation Protocols
7
Enzymatic Dissociation of hCS-FF and hCS/hSS
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For the enzymatic dissociation of hCS-FF for culture in monolayer and
immunocytochemistry (ani-GFAP), up to 3 spheroids were incubated with 200
μl of Accutase (Innovative Cell Technologies) for 20 min at 37 °C,
washed with neural medium and gently triturated using a P200 pipette. Cells were
plated on glass coverslips (15 mm, Warner Instruments) coated with
poly-L-ornithine and laminin (Sigma-Aldrich) at a density of around 250,000
cells per coverslip in neural medium supplemented with BDNF, NT3 and, for the
first 24 hours, 10 μM Y-27632.
To dissociate hCS and hSS for single-cell profiling, we used a
previously published protocol7 (link).
Briefly, 3–10 spheroids were chopped using a #10 blade and then incubated
in 40 U/ml papain enzyme solution containing 0.46% D(+)-Glucose (Sigma-Aldrich),
26 mM NaHCO3 (Sigma-Aldrich), 0.5 mM EDTA (Sigma-Aldrich) in EBSS
(1X, Sigma-Aldrich) at 37 °C in 5% CO2 for 70 minutes. The
digested spheroids were then washed and carefully triturated with a protease
inhibitor stock solution containing 0.46% D(+)-Glucose (Sigma-Aldrich), 26 mM
NaHCO3 (Sigma-Aldrich), 5 mg trypsin inhibitor (Sigma-Aldrich) in
EBSS (1X, Sigma-Aldrich). After centrifugation (200 × g for 4 min), the
pellet was resuspended in 0.2% bovine serum albumin (BSA) diluted in PBS and
supplemented with 10 μ M Y-27632, and the cells were used for the
single-cell RNA sequencing.
immunocytochemistry (ani-GFAP), up to 3 spheroids were incubated with 200
μl of Accutase (Innovative Cell Technologies) for 20 min at 37 °C,
washed with neural medium and gently triturated using a P200 pipette. Cells were
plated on glass coverslips (15 mm, Warner Instruments) coated with
poly-L-ornithine and laminin (Sigma-Aldrich) at a density of around 250,000
cells per coverslip in neural medium supplemented with BDNF, NT3 and, for the
first 24 hours, 10 μM Y-27632.
To dissociate hCS and hSS for single-cell profiling, we used a
previously published protocol7 (link).
Briefly, 3–10 spheroids were chopped using a #10 blade and then incubated
in 40 U/ml papain enzyme solution containing 0.46% D(+)-Glucose (Sigma-Aldrich),
26 mM NaHCO3 (Sigma-Aldrich), 0.5 mM EDTA (Sigma-Aldrich) in EBSS
(1X, Sigma-Aldrich) at 37 °C in 5% CO2 for 70 minutes. The
digested spheroids were then washed and carefully triturated with a protease
inhibitor stock solution containing 0.46% D(+)-Glucose (Sigma-Aldrich), 26 mM
NaHCO3 (Sigma-Aldrich), 5 mg trypsin inhibitor (Sigma-Aldrich) in
EBSS (1X, Sigma-Aldrich). After centrifugation (200 × g for 4 min), the
pellet was resuspended in 0.2% bovine serum albumin (BSA) diluted in PBS and
supplemented with 10 μ M Y-27632, and the cells were used for the
single-cell RNA sequencing.
8
Cardiac Myocytes from Human iPSCs
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Commercially available human male and female iPSC-derived cardiac myocytes (iPS-CMs, Axol Bioscience, UK) were prepared according to the manufacturer’s instructions. Briefly, cardiac myocytes (1 ml, stored frozen in liquid N2) thawed in a 37 °C water bath were initially suspended in 1 ml Axol Complete Cardiomyocyte Medium (warmed to 37 °C). An additional 8 ml of medium was added and mixed by gentle inversion. Cardiac myocytes were seeded (25,000 cells/well) in 1.5 ml medium in 12 well culture plates that contained glass coverslips (15-mm diameter, Warner Instruments, CT) pre-coated with Matrigel™ (Corning Life Sciences, MA). Cells were incubated for 24 h at 37 °C, 7% CO2. Non-adherent cells were removed by rinsing with media. After 7 days in culture, 17-β-estradiol (Sigma) was added to the media at a final concentration of 1 nM. The iPS-CMs were kept in 17-β-estradiol for 24–48 h before measuring ICa,L or INCX by voltage-clamp technique. Control cells were treated with media containing DMSO (< 0.1%). Cultured iPS-CMs had a tendency to mature and exhibit spontaneous contractions by the 7th day of incubation. The male and female iPS-CMs used in this study had common features found in all iPS-CMs: spontaneous automaticity and contractions and stable ionic currents for weeks, as previously reported [34 (link)].
9
HEK 293 Cell Culture and Plating
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HEK 293 cells (ATCC CRL 1573, Rockville, MD, USA; hereafter HEK cells) were maintained in humidified 5% CO2 (at 37 °C) in Dulbecco's modified Eagle's medium with GlutaMax (Invitrogen, Carlsbad, CA, USA) supplemented with 10% dialysed fetal bovine serum, 10 μg ml−1 streptomycin, and 10 units ml−1 penicillin, as previously described (Yuan et al. 2009 ). Cells were trypsinized and plated onto glass coverslips (Warner Instruments, Hamden, CT, USA) coated with poly‐d ‐lysine at 100 μg ml−1 for single channel and 5 μg ml−1 for whole‐cell recordings. Coverslips were transferred to 24‐well plates with 0.5 ml of supplemented media in each well.
10
Piezo1, TREK1, and TRAAK Overexpression in HEK293T Cells
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HEK293T cells (ATCC #11268 authenticated and tested mycoplasma free, Manassas, VA) were cultured at 37 °C and 5% CO2 in DMEM-HG (Life Technologies, Carlsbad, CA) supplemented with 10% FBS (Clontech, Mountain View, CA), 50 U/mL penicillin, and 50 mg/mL streptomycin (Life Technologies). Cells were reseeded into 6-well plates and transfected (mouse Piezo1-pIRES-EGFP (70 (link)), mouse TREK1-pIRES2-EGFP, and mouse pCEH-TRAAK-GFP, YFP alone: 3 µg; mouse Piezo2 (54 (link)): 2.5 µg + 0.5 µg YFP) with FuGENE 6 (Promega, Madison, WI) 48 h prior to recording at a transfection ratio of 10 µL FuGENE 6:3 µg total DNA. Glass coverslips (1.5 mm; Warner Instruments, Hamden, CT) were coated with 0.1 mg/mL poly-L-lysine at room temperature for 30 min followed by 1 µg/mL laminin at 37 °C for 45 min to 1 h. Cells were trypsinized and transferred onto coated coverslips16 to 24 h before recording. Cell cultures were limited to 20 passages.
Cells were treated with cytochalasin D (10 μM solubilized in dimethyl sulfoxide (DMSO); both Sigma, Burlington, MA) for 1 h prior to and throughout the recording session. Control cells were day-matched and treated with an equal volume of DMSO over the same time course.
Cells were treated with cytochalasin D (10 μM solubilized in dimethyl sulfoxide (DMSO); both Sigma, Burlington, MA) for 1 h prior to and throughout the recording session. Control cells were day-matched and treated with an equal volume of DMSO over the same time course.
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