The BTE technology used in the present study included two main elements: a biodegradable PDLLA polymer membrane (Hong Jian Bio-Medical Products Co., Ltd, Guangzhou, China, http://www.hongjianbio.com ) and BMSCs. Briefly, 5.0 × 4.0 × 0.4-mm membranes were rinsed in sterile normal saline three times and in LG-DMEM twice, and then laid in the bottoms of wells of six-well plates. Next, 500-µL amounts of medium containing 5 × 106 third-generation BMSCs/mL were seeded into each well, and the plates were incubated at 37° C under 5% (v/v) CO2, with cell growth status being checked daily. After 5 days’ incubation, the BTE complexes were examined by Leica TCS SP5 confocal microscopy (Leica, Mannheim, Germany).
Tcs sp5 confocal microscopy
Manufactured by Leica
Sourced in Germany
The Leica TCS SP5 is a confocal microscopy system designed for high-resolution imaging. It features a fully-motorized scan head and is capable of acquiring images with high contrast and signal-to-noise ratio. The system is equipped with multiple lasers, allowing for the visualization of a wide range of fluorophores. It supports a variety of imaging techniques, including confocal, multiphoton, and super-resolution microscopy.
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Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (3 mentions)
Anti hrp tritc, by Jackson ImmunoResearch (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Methylbutane, by Merck Group (2 mentions)
Rat anti mouse cd16 cd32, by BD (2 mentions)
Polyclonal guinea pig anti insulin antibody, by Agilent Technologies (2 mentions)
Alexafluor 647 conjuaged goat anti rabbit antibody, by Thermo Fisher Scientific (2 mentions)
Alexafluor 555 conjugated anti guinea pig antibody, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 conjugated anti mouse antibody, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 488 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Oct media, by Sakura Finetek (1 mentions)
Hoechest 33342, by Thermo Fisher Scientific (1 mentions)
Axio observer a1 fluorescence microscope, by Zeiss (1 mentions)
Normal donkey serum (nds), by Merck Group (1 mentions)
Dihydroethidium dhe staining, by Merck Group (1 mentions)
Las af lite software, by Leica (1 mentions)
Tissue tek oct embedding medium, by Sakura Finetek (1 mentions)
Cy5 dutp, by GE Healthcare (1 mentions)
Chromatide alexa fluor 594 5 dutp, by Thermo Fisher Scientific (1 mentions)
24 protocols using tcs sp5 confocal microscopy
1
PDLLA-BMSC Construct for Bone Tissue Engineering
2
Immunofluorescence Imaging of BK Protein
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BK knockout clones and wild type cells were seeded on cell slides. At 70% confluent, cells were fixed in 4% paraformaldehyde for 15 min. Following washes with PBS, cells were permeabilized in 0.1%Triton X-100 for 10 min and blocked in 3% bovine serum albumin (BSA) in PBS buffer for 1 h at room temperature. The primary antibody for BK protein (1:200, Abcam, UK) was incubated overnight at 4°C. The secondary AlexaFluor647-conjugated anti-mouse antibody (Jackson ImmunoResearch, USA) was used at a 1:200 dilution for 1 hour at room temperature. Images were captured using a Leica TCS SP5 confocal microscopy (Leica, Germany)
3
Visualizing Neuronal and Glial Markers
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NMJs from third instar larvae and adult brains from flies of 1 week old without mouthparts were dissected and fixed with 4% formaldehyde in 1X PBS following standard protocols previously described. Reagents used include: Normal Donkey Serum (0.5%, NDS, Jackson Lab), mouse anti-Repo (1:100, DSHB), rat anti-Elav (1:500, DSHB), rabbit anti-Cnx (1:500, gift from Nansi Colley), mouse anti-Para (1:100, made in this study), anti-HRP-TRITC (1:500, Jackson Lab), and anti-HRP-Cy5 (1:500, Jackson Lab). All other secondary antibodies were purchased from Jackson Lab. Images were captured with Leica TCS SP5 confocal microscopy. Details on brain dissection, immunostaining, image acquisition, and quantification of fluorescent intensities were included in the supplementary materials.
4
Quantifying Retinal Photoreceptor Changes
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To assess how the treatment affected retinal photoreceptors (i.e., thickness of the ONL), we used eyes from each group (n ¼ 4 or 5). Eyes were enucleated on D90 and fixed with 2% paraformaldehyde, cryoprotected, and embedded in optimum cutting temperature media (OCT; Sakura Finetek, Torrance, CA, USA) as previously described. 34 We crosssectioned whole eyes and obtained 20 sections (28 lm apart), including those containing the inferior pole, optic nerve, and superior pole. Briefly, 14-lm-thick cryosections were stained using TO-PRO-3 iodide (T3605, 1:10,000; Thermo Fisher Scientific, Waltham, MA, USA). Samples were examined by Leica TCS SP5 confocal microscopy (Leica). ONL measurements were made at different regions around the entire retinal section (center, middle, and periphery). ImageJ software was used to calculate the ONL thickness at each location, and the mean ONL thickness of the entire retina was compared among the different groups.
5
Quantifying Retinal C3 Expression
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To inspect how the treatment affected complement component 3 (C3), eyes (n ¼ 4 or 5) from each group were enucleated on D90 (same eyes used in the nuclear staining) and fixed, cryoprotected, and embedded in OCT media (Sakura Finetek). Whole eyes were cross-sectioned as previously described and incubated with a mouse monoclonal IgG 1 C3 antibody (1:500, Santa Cruz Biotechnology, Dallas, TX, USA) followed by Alexa Fluor 488 donkey antimouse IgG (1:200; Invitrogen, Carlsbad, CA, USA). The slides were examined by Leica TCS SP5 confocal microscopy (Leica).
6
Immunofluorescence Assay for SARS-CoV-2 Antibodies
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After infection, AC16 and Vero cells were fixed in 4% formaldehyde for 1 h at room temperature after infection. Cells were permeabilized in 0.5% Triton X‐100 and blocked in 5% BSA in PBS. The cells were then probed with the serum of the patient or healthy control at a dilution of 1:1000 for 1 h at room temperature. After three times of wash with PBS, cells were incubated with goat anti‐human IgG antibody conjugated with Alexa fluor 488 at a dilution of 1:1000 for 1 h (Invitrogen). The cells were then washed and stained with Hoechest‐33342 (Invitrogen) to detect nuclei. Fluorescence images were acquired by a TCS SP5 confocal microscopy (Leica Microsystems).
7
Cellular Uptake Kinetics of PZL318
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HeLa or HaCaT cells (5×104 cells/mL) were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM) [containing 10% (v/v) fetal calf serum, 60 μg mL−1 penicillin, and 100 μg mL−1 streptomycin] at 37°C in a humidified atmosphere containing 5% CO2 for 24 hours. After removing the original medium, a solution of PZL318 (final concentration: 40 μM, in complete DMEM medium as above; 1 mL/well) was added, and cells were incubated at 37°C in a humidified atmosphere (containing 5% CO2) for 0 minutes, 15 minutes, 30 minutes, 60 minutes, 120 minutes, and 360 minutes. After removing the medium, the cells were washed with fresh medium (1 mL ×3 times) and resuspended in fresh medium (1 mL/well). PZL318 on the cell surface and in the cytoplasm were analyzed by use of an Axio Observer A1 fluorescence microscope (Carl Zeiss Meditec AG, Jena, Germany) or TCS SP5 confocal microscopy (Leica Microsystems, Wetzlar, Germany), respectively.
8
Immunofluorescence Staining of hiPSC-RPE Cells
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hiPSC-RPE cells were fixed in 4% paraformaldehyde for 20 min at room temperature, permeabilized, and blocked with tris-buffered saline (TBS) containing 0.5% (v/v) Triton X-100 and 6% (v/v) normal donkey serum (Sigma-Aldrich; St. Louis, MO, USA) for 1 h at room temperature. Primary antibodies were incubated at 4 °C overnight in phosphate-buffered saline (PBS), 0.1% (v/v) Triton X-100, and 6% (v/v) normal donkey serum, and secondary antibodies (1:500; Jackson Immunoresearch Laboratories, West Grove, PA, USA) were incubated in the same buffer at 37 °C for 2 h. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich; St. Louis, MO, USA) and cells were mounted in an anti-fading mounting medium. Confocal images were obtained on a TCS SP5 confocal microscopy (Leica Microsystems, Wetzlar, Germany) using a 40× or 63× oil immersion objective, and z-stacks were acquired at 1024 × 1024 pixels. Antibodies are listed in Supplementary Table S1 .
9
Quantifying Superoxide Levels in Mouse Hearts
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In situ detection of superoxide anion (O2−) levels was performed in mouse heart sections by dihydroethidium (DHE) staining (Sigma–Aldrich), as previously described [24 (link)]. Heart samples were embedded in Tissue Tek OCT embedding medium (Sakura Finetek Europe B.V., Alphen aan den Rijn, The Netherlands), and frozen in liquid nitrogen. Cardiac slices were equilibrated for 30 min at 37 °C in Krebs-HEPES buffer (in mmol/L: NaCl 130, KCl 5.6, CaCl2 2, MgCl2 0.24, HEPES 8.3, and glucose 11, pH = 7.4), incubated with DHE (2 μM), cover-slipped and maintained for 30 min in a humidified chamber at 37 °C in the dark. The fluorescent signal was analyzed with a fluorescent laser scanning confocal microscope (Leica TCS SP5 confocal microscopy (20× for quantification and illustration) and the Leica LAS AF Lite software (Leica Microsystems S.L.U). Fluorescent images were acquired using a 561 nm laser (long-wavelength excitation). Data were expressed as % of signal in control samples to minimize laser fluctuations from one day to another.
10
3D FISH Analysis of Igκ Locus
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