Cells were fixed in 4% PFA in phosphate-buffered saline (PBS) at room temperature (RT) for 30 min and blocked with 3% bovine serum albumin in PBS (3% B-PBS). Cells were incubated with primary antibodies in 3% B-PBS at 4 °C overnight. Secondary antibodies conjugated to Alexa Fluor 594 (Thermofisher) or Alexa Fluor 488 (Thermofisher) in 3% B-PBS were added and incubated at RT for 1 hr, followed by several washes with PBS. The staining with transferrin-594 conjugate (Thermofisher) was performed following the manufacturer’s protocol. For HLC cultures grown on transwell filters, the filter was removed from the hanging insert and submerged, cell side up, in PBS using a slice anchor (Warner Instruments). Cultures were imaged on an Upright BX61WI microscope (Olympus) with a UMPlan FL 60 × 1.0 NA water dipping objective (Olympus) or UMPlan FL ×10, 0.3 NA water dipping objective and an Orca Flash 4.0 digital CMOS camera (Hamamatsu) using MetaMorph image acquisition software (Molecular Devices). Deconvolution was performed using the standard adaptive point spread settings in Autoquant (Media Cybernetics). Image analysis was conducted using Fiji.
Slice anchor
Manufactured by Warner Instruments
Sourced in United States
The Slice Anchor is a versatile lab equipment item designed for securing samples during precision cutting or slicing operations. It provides a stable platform to hold specimens in place, enabling accurate and reproducible sectioning.
Automatically generated - may contain errors
Lab products found in correlation
Autoquant, by Media Cybernetics (1 mentions)
Metamorph image acquisition software, by Molecular Devices (1 mentions)
Orca flash4.0 digital cmos camera, by Hamamatsu Photonics (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Bx61wi upright microscope, by Olympus (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Umplan fl 60 1.0 na water dipping objective, by Olympus (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Soybean trypsin inhibitor, by Merck Group (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Discovery v8, by Zeiss (1 mentions)
Ames media, by Merck Group (1 mentions)
Vt1200, by Leica (1 mentions)
Igor pro 6, by Wavemetrics (1 mentions)
Ames medium, by Merck Group (1 mentions)
6 protocols using slice anchor
1
Immunofluorescence Imaging of Liver Cells
2
Live Imaging of Tumor Slices
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Freshly excised tumor slices were obtained from KPCT and KPCG mice. Live cell imaging of tumor slices was performed similar to time-lapse MPE methods described previously (27 (link)). Briefly, the tumor was sliced into a few thin sections (300–350 µm) using a vibratome, set on a 35 mm dish with a slice anchor (Warner Instruments), and overlaid with L-15 media supplemented with 10% FBS, penicillin-streptomycin, plasmocin, fungizone, and 10 μg/mL soybean trypsin inhibitor (MilliporeSigma). Imaging was subsequently performed on the multiphoton microscopy setup described above with a custom-built temperature-controlled stage insert (60 (link)) for 6–12 hours with a time interval of 20 minutes between frames at 880–900 nm excitation wavelength and emission captured in the green (KPCG), red (KPCT), or blue (SHG) channels. Analysis and 3D rendering of Z stacks, visualization of time-lapse imaging data, and image processing were done in Fiji. For live imaging, Z stacks were generally obtained at a step size of 4–5 μm, covering a total depth of about 80–100 μm.
3
Methacholine-Induced Bronchoconstriction Assay
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Bronchoconstriction was measured according to the protocol by Seehase et al. [21 ]. One slice was transferred into a 6-well plate with 2 mL DMEM (supplemented with 25 mM HEPES) per measurement and fixed with a slice anchor (Warner Instruments, Hamden, CT, USA). Methacholine (Mch) was added stepwise. After addition of each concentration of Mch (10−9 to 10−3 M), pictures were recorded in 5 s intervals for 3 min with a stereo microscope (Discovery V8; Zeiss, Jena, Germany) controlled by the Axio Vision 4.8.2. software program (Zeiss, Jena, Germany). Images were analyzed with the ImageJ analysis program (National Institute of Health).
4
Retinal Preparation for Light Response Studies
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For experiments that measured light responses, mice were dark-adapted overnight before killing them with CO2 inhalation followed by cervical dislocation. Retinas were isolated under dim red light and retinas were incubated in a solution containing collagenase and hyaluronidase dissolved in oxygenated (95% O2 and 5% CO2) Ames media (Sigma-Aldrich) at room temperature 15–30 min to aid in penetration of the inner limiting membrane (Schmidt and Kofuji, 2011 (link)). Retinas were then mounted in the recording chamber and held in place with a slice anchor (Warner Instruments) and perfused with Ames bubbled with 95% O2 and 5% CO2 at a rate of 4–6 ml/min. For optogenetic and puffing experiments, mice were dark-adapted for 1 h before sacrifice, and all manipulations were carried out in room light.
5
Acute Brain Slice Preparation and Electrophysiology
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Mice were deeply anesthetized and perfused transcardially with ice-cold ACSF (pH 7.3-7.4) containing (in mM): 124 NaCl, 2.5 KCl, 1.2 NaH2PO4, 24 NaHCO3, 5 HEPES, 12.5 glucose, 1.3 MgSO4, 7H2O, 2.5 CaCl2. The brain was rapidly removed, and coronal sections (250uM) unless otherwise indicated, were cut on a vibratome (VT1200s, Leica).
Slices were incubated in a holding chamber for 12-15 minutes at 32-34°C in a NMDGbased recovery solution (pH 7.3-7.4, pH adjusted with HCl) (in mM): 92 NMDG, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 5 sodium ascorbate, 2 thiourea, 3 sodium pyruvate, 10 MgSO4, 7H2O, 0.5 CaCl2. Osmolarity for the NMDG-based solution and ACSF was kept between 300-310 mOsm. Following incubation, slices were moved to a second holding chamber containing ACSF at room temperature (20-22°C) for at least 1 hr. prior to recording. For recording, slices are transferred to the recording chamber (Scientifica) fully submerged in oxygenated (95% O2, 5% CO2) ACSF at a perfusion rate of 1.4-1.6 mL/min, bath temperature of 29-30°C, and secured using a slice anchor (Warner Instruments). Electrophysiology data were acquired using custom-built
Recording Artist software version (Rick Gerkin), Igor Pro 6.37 (Wavemetrics). All recordings were sampled at 20kHz, filtered at 2.8kHz except for the in vivo modeled trains that were sampled at 10kHz.
Slices were incubated in a holding chamber for 12-15 minutes at 32-34°C in a NMDGbased recovery solution (pH 7.3-7.4, pH adjusted with HCl) (in mM): 92 NMDG, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 5 sodium ascorbate, 2 thiourea, 3 sodium pyruvate, 10 MgSO4, 7H2O, 0.5 CaCl2. Osmolarity for the NMDG-based solution and ACSF was kept between 300-310 mOsm. Following incubation, slices were moved to a second holding chamber containing ACSF at room temperature (20-22°C) for at least 1 hr. prior to recording. For recording, slices are transferred to the recording chamber (Scientifica) fully submerged in oxygenated (95% O2, 5% CO2) ACSF at a perfusion rate of 1.4-1.6 mL/min, bath temperature of 29-30°C, and secured using a slice anchor (Warner Instruments). Electrophysiology data were acquired using custom-built
Recording Artist software version (Rick Gerkin), Igor Pro 6.37 (Wavemetrics). All recordings were sampled at 20kHz, filtered at 2.8kHz except for the in vivo modeled trains that were sampled at 10kHz.
6
Rabbit Retina Electrophysiology Protocol
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In vitro experiments were conducted at the University of New South Wales (UNSW) on NZ White rabbit (2.5-3.5 kg) retina preparations. In total, data from 7 cells were collected, some of which were repeatedly stimulated with the stimulation protocols described below.
All procedures were approved by the UNSW Animal Care and Ethics Committee. To anaesthetize the rabbits, Isoflurane (4 % in O 2 , 2 L/min) was delivered through a mask. Subsequently, one eye was enucleated and the animal was immediately euthanized by lethal overdose of sodium pentobarbital (150 mg/kg). The eye was placed in sodium bicarbonate buffered (pH 7.4) Ames medium (Sigma Aldrich), which was continuously bubbled with 95 % O 2 and 5 % CO 2 at room temperature. A 5 mm by 10 mm piece of inferior retina with attached sclera, including the optic nerve head, was excised. The tissue was placed flat, RGC side up, in a perfusion chamber (Warner instruments) and was held in place using a slice anchor (Warner Instruments). It was perfused at a rate of 4-5 mL/min with oxygenated Ames medium (pH 7.4, 36degC).
All procedures were approved by the UNSW Animal Care and Ethics Committee. To anaesthetize the rabbits, Isoflurane (4 % in O 2 , 2 L/min) was delivered through a mask. Subsequently, one eye was enucleated and the animal was immediately euthanized by lethal overdose of sodium pentobarbital (150 mg/kg). The eye was placed in sodium bicarbonate buffered (pH 7.4) Ames medium (Sigma Aldrich), which was continuously bubbled with 95 % O 2 and 5 % CO 2 at room temperature. A 5 mm by 10 mm piece of inferior retina with attached sclera, including the optic nerve head, was excised. The tissue was placed flat, RGC side up, in a perfusion chamber (Warner instruments) and was held in place using a slice anchor (Warner Instruments). It was perfused at a rate of 4-5 mL/min with oxygenated Ames medium (pH 7.4, 36degC).
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