HEC-1A cells were obtained from Servicebio (Wuhan, China). The CRISPR/Cas9 plasmid was purchased from GenePharma (Shanghai, China). The plasmid extraction kit and reverse transcription (RT)-PCR kit were obtained from Omega Bio-tek (Norcross, GA, USA). High-glucose Dulbecco’s modified Eagle’s medium (DMEM) with serum phosphate-buffered saline (PBS) and fetal bovine serum (FBS) were purchased from HyClone (Jülich, Germany). Dipalmitoylphosphatidylethanolamine (DPPE) and dipalmitoylphosphatidylcholine (DPPC) were obtained from Lipoid (Steinhausen, Switzerland). DC-cholesterol (3ß-[N-(N′, N′-dimethylaminoethane)-carbamoyl]cholesterol) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). The IX51 inverted fluorescence microscope was obtained from Olympus (Tokyo, Japan). The GCZZ ultrasound transfection system was developed by Chongqing Medical University (Chongqing, China). The 7500 Real-Time PCR System was from Applied Biosystems (Foster City, CA, USA).
Ix51 inverted fluorescence microscope
Manufactured by Olympus
Sourced in Japan, United States
The IX51 inverted fluorescence microscope is a high-performance optical instrument designed for a variety of imaging applications. It features a sturdy, inverted design and supports a range of fluorescence techniques, enabling detailed analysis and observation of samples.
Automatically generated - may contain errors
Lab products found in correlation
Cellsens software, by Olympus (4 mentions)
H 800 electron microscope, by Hitachi (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Hec 1a cells, by Wuhan Servicebio Technology (1 mentions)
Dipalmitoylphosphatidylcholine dppc, by Lipoid (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Plasmid extraction kit, by Omega Bio-Tek (1 mentions)
Sudan black solution, by Merck Group (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Fura 2, by Olympus (1 mentions)
Fura 2 am, by Thermo Fisher Scientific (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Ifn γ, by Merck Group (1 mentions)
Dp30bw digital camera, by Olympus (1 mentions)
Fc500 flow cytometry system, by Beckman Coulter (1 mentions)
Optima 3300 dv, by PerkinElmer (1 mentions)
Zetasizer nano z, by Malvern Panalytical (1 mentions)
Uv 2600 spectrophotometer, by Shimadzu (1 mentions)
X ray diffractometer, by Rigaku (1 mentions)
Paraformaldehyde (pfa), by Santa Cruz Biotechnology (1 mentions)
34 protocols using ix51 inverted fluorescence microscope
1
CRISPR/Cas9 Transfection in HEC-1A Cells
2
Apoptosis and Necrosis Quantification
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Costaining with Hoechst33342 and PI was performed to evaluate the mode of cell death. After treatment, H460 cells at a density of 1 × 104 cells/well in 96-well plates were incubated with 10 µM Hoechst33342 and 5 µg/mL PI for 30 min at 37 °C. The cells were washed and then observed under an Olympus IX51 inverted fluorescence microscope (Olympus, Tokyo, Japan). Bright blue fluorescence of Hoechst33342 declares chromatin condensation and/or nuclei fragmentation in apoptotic cells, while the red fluorescence of PI indicates necrotic cell death [55 (link)]. The relative number of apoptotic cells to the total cell from three different observed areas was calculated and presented as the percentage apoptosis.
3
Imaging Vesicular Zinc in Hippocampal Slices
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Acute hippocampal slices were prepared as described in the supplementary information. Slices were incubated with 6 µM of BP for 30 min at room temperature in PFS, continuously stirred with a jet of 95% O2 and 5% CO2. Cell-permeable form of dyes Fluozin-3 AM and TSQ (N-(6-Methoxy-8-Quinolyl)-p-Toluenesulfonamide) were used as positive controls for vesicular Zn2+ detection. Fluozin-3 AM (Kd (Zn2+) ~15 nM) is suitable for detection of Zn2+ concentrations in the 1–100 nM range. Fluozin-3 AM was incubated at a concentration of 2.5 µM in PFS for 15 min. TSQ is also selective for zinc in the presence of physiological concentrations of Ca2+ and Mg2+, and was incubated at a concentration of 90 µM for 20 min. The stained slices were washed with PFS to remove excess dye and observed under Olympus IX51 inverted fluorescence microscope at an excitation/emission wavelength of 460–490/520 nm for BP, Fluozin-3 AM and 330–385/420 nm for TSQ respectively. Fluozin-3 AM stained slices were imaged only after 30 min to allow the AM ester to cleave inside the cells for fluorescence emission.
4
Double Immunofluorescence for TREM2 and DAP12
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We also conducted double immunofluorescence to determine the relationship between TREM2 and DAP12 expression in the brain tissues. A sequential labeling procedure was applied. We first processed the immunolabeling of TREM2 with primary antibody as described earlier, followed by biotinylated anti‐goat immunoglobulin G (IgG) and streptavidin‐conjugated Alexa 488 (Molecular Probe, Life Technologies, Carlsbad, CA, USA). After completion of the first round of immunolabeling, we applied the primary antibody of DAP12 overnight, followed by 2‐hour incubation with anti‐rabbit IgG (H + L) conjugated with Alexa 568. Both fluorescence‐dye‐conjugated antibodies were used at 1:2000 from the stock provided by the manufacturer. To reduce nonspecific autofluorescence background from human brain tissues, mounted tissue sections were immersed in 1% Sudan Black solution (Sigma‐Aldrich, St. Louis, MO, USA) in 70% alcohol for 5 minutes. The excessive Sudan Black was removed by brief immersion of the slides in 70% alcohol and distilled water before coverslipping 26 . Slides were coverslipped with Vectashield (Vector Laboratories, Burlingame, CA, USA). The images were captured with a charge‐coupled device (CCD) camera attached to the Olympus IX51 inverted fluorescence microscope (Olympus, Center Valley, PA, USA). Image overlay was composed by the Olympus software DP Controller version 3.2.1.276.
5
Evaluating Cisplatin and S68345 Efficacy in Breast Cancer Cells
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MDA-MB-468 and MDA-MB-231 cells were seeded in a 6-well plate in RPMI 1640 (1×) supplemented with 2.5% FBS at a density of 1 × 104 cells/well. Twenty-four hours post treatment, cells were treated with various concentrations of cisplatin and S68345 and incubated in a humidified atmosphere of 5% CO2 at 37 °C for 6 days. Medium was removed, and cells were washed with 1× dPBS fixed with 4% formaldehyde at 4 °C for 30 min while rocking. Cells were washed with 1× dPBS and incubated in 0.5% crystal violet at room temperature for 1 h. Staining solution was removed, and cells were washed 4 times with 1× dPBS. Cells were imaged on an Olympus IX51 Inverted Fluorescence microscope (Olympus, Tokyo, Japan). Images were quantified using Fiji Image J (imagej.net; Version 1.0) where colonies >5 cells were considered for analysis.
6
Characterization of PAA-Coated Gold Nanorods
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A Ultraviolet (UV)-1800 UV-vis-NIR spectrophotometer (Shimadzu, Japan) was used
to detect the spectra of UV visible absorption of all samples for GNRs@PAA
preparation ranging from 400 nm to 1100 nm wavelength in common experimental
environment with room temperature. Transmission electron microscopy (TEM) images
of PAA-coated GNRs were implemented by a Hitachi H-800 electron microscope
(Japan) under 200 kV accelerating voltage with a charge coupled device camera.
Fourier transform infrared (FTIR) spectra were measured using an IR Affinity-1
spectrophotometer (Avatar, Nicolet, USA). Bright-field and fluorescent mappings
of MG63 cells were obtained using an Olympus IX51 inverted fluorescence
microscope (Olympus, Japan).
to detect the spectra of UV visible absorption of all samples for GNRs@PAA
preparation ranging from 400 nm to 1100 nm wavelength in common experimental
environment with room temperature. Transmission electron microscopy (TEM) images
of PAA-coated GNRs were implemented by a Hitachi H-800 electron microscope
(Japan) under 200 kV accelerating voltage with a charge coupled device camera.
Fourier transform infrared (FTIR) spectra were measured using an IR Affinity-1
spectrophotometer (Avatar, Nicolet, USA). Bright-field and fluorescent mappings
of MG63 cells were obtained using an Olympus IX51 inverted fluorescence
microscope (Olympus, Japan).
7
DNA Methylation Profiling in Zygotes
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Zygotes were fixed in 3.7% paraformaldehyde in PBS for 30 min at room temperature and permeabilized for 15 min in 0.1% Triton X-100 in PBS at room temperature. 5mC and 5-hydroxymethylcytosine (5hmC) staining was carried out as previously described (33 (link)). The antibody information is provided in Supplementary Table S2 . Images were taken with Olympus IX51 inverted fluorescence microscope.
8
Measurement of Intracellular Calcium Dynamics
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Intracellular Ca2+ concentration measurements were obtained from PTC of WT and TRPC6-/- mice preloaded with the Ca2+-sensitive fluorescent dye Fura2-AM (Invitrogen, F1201, USA). As described in He et al.41 (link), PNAS 2017, briefly, the cells were loaded with 3 μM Fura2-AM in DMEM/F12 1:1 medium for 50 min at room temperature. Then the cells were washed 3 times with HBSS (140 mM NaCl, 5 mM KCl, 10 mM HEPES, 10 mM glucose, and 1 mM MgCl2, pH 7.4) medium with 2 mM Ca2+ and incubated at room temperature for another 10 min. The coverslips were mounted onto the platform of an inverted epifluorescence microscope. To measure Thapsigargin (Tg, Invitrogen, T7459, USA)-evoked Ca2+ entry, cells were bathed in sequence with 50 μM EGTA in HBSS for 3 min, 50 μM EGTA and 2 μM Tg in HBSS for 6 min, and 2 mM Ca2+ plus 2 μM Tg in HBSS for 6 min, as shown in the figures. Ca2+ entry was also assessed in the absence and presence of the TRPC inhibitor SAR7334. Cytosolic Ca2+ was monitored with an Olympus IX51 inverted fluorescence microscope and SlideBook software, using excitation wavelengths of 340 and 380 nm to detect Fura-2/Fura2-Ca2+ fluorescence emissions at 510 nm.
9
Cardiomyocyte Calcium Imaging with Fluo-4 AM
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Cardiomyocytes were washed with Tyrode solution consisting of 140 mM NaCl, 5 mM KCl, 5 mM HEPES, 1 mM NaH2PO4, 1 mM MgCl2, 1.8 mM CaCl2 and 10 mM glucose (pH 7.4). Cells were loaded with 0.01 mM Fluo-4 AM dye (Life Technologies—Molecular Probes, F14201) in darkness at room temperature (RT) for 1h. Following dye loading, the cells were washed with Tyrode solution and further incubated for 20 min at RT. Imaging was performed on an Olympus IX-51 inverted fluorescence microscope with an Olympus SP-500UZ camera. The beating frequency and the width of individual peaks were analyzed from recordings by a CBAnalyser as described previously [30 (link)].
10
Cytosolic Calcium Dynamics Measurement
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Calcium imaging was performed as previously described (Hou et al., 2018 (link)). Briefly, the cells were loaded with 3 μM Fura2-AM in a DMEM/F12 medium at 1:1 for 50 min at room temperature. Then, the cells were washed three times with an HBSS medium (140 mM NaCl, 5 mM KCl, 10 mM HEPES, 10 mM glucose, and 1 mM MgCl2, pH 7.4) with 2 mM Ca2+ and incubated at room temperature for another 10 min. The coverslips were mounted onto the platform of an inverted epifluorescence microscope. Cytosolic Ca2+ was monitored with an Olympus IX51 inverted fluorescence microscope and SlideBook software, using excitation wavelengths of 340 and 380 nm to detect Fura-2/Fura2-Ca2+ fluorescence emissions at 510 nm.
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