After the cells were fixed with 4% formaldehyde for 15 min, the membranes were broken with 0.2% Triton X-100 for 5-10 min. Then, samples were incubated with 3% BSA at room temperature for 60 min. Soon afterward, they were incubated overnight at 4°C with primary antibodies in blocking buffer. After washing with TBST three times, the samples were incubated with 1 : 800 secondary antibody-labeled fluorescent for 60 min. The nucleus was then stained with DAPI for 5 min. After washing with PBS three times, fluorescence images were taken with confocal microscopy (Smartproof 5, Carl Zeiss, Oberkochen, Germany). 10 visual fields were collected for further analysis of each image. Differently, after the ROS reagent was directly added to the sample according to the protocol, it was immediately observed under a confocal microscope (Smartproof 5, Carl Zeiss, Oberkochen, Germany), and the ImageJ software (NIH, Bethesda, MD, USA) was used for quantitative analysis.
Smartproof 5
Manufactured by Zeiss
Sourced in Germany
Smartproof 5 is a 3D optical surface metrology system developed by ZEISS. It is designed to capture and analyze the surface topography of a wide range of materials and components with high accuracy and resolution.
Automatically generated - may contain errors
Lab products found in correlation
Zen smartproof hf2 1, by Zeiss (2 mentions)
Confomap 7, by Zeiss (2 mentions)
Confomap st 7, by Zeiss (2 mentions)
Nunc petri dish, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Geminisem 500, by Zeiss (1 mentions)
Dapi fluoromount g, by Zeiss (1 mentions)
S 4800 microscope, by Hitachi (1 mentions)
Squid vsm, by Quantum Design (1 mentions)
Lambda 950, by PerkinElmer (1 mentions)
D8 discover, by Bruker (1 mentions)
Cypher s, by Oxford Instruments (1 mentions)
Confomap software, by Zeiss (1 mentions)
15 protocols using smartproof 5
1
Immunofluorescence and ROS Imaging Protocol
2
Immunofluorescence Staining of LC3 in A549 Cells
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A549 cells cultivated in a Nunc™ Petri dish (Thermo Fisher Scientific, Inc.) were washed three times with 1X PBS, fixed in 4% paraformaldehyde for 20 min at 4˚C and washed again three times with PBS. The cells were then permeabilized with 0.2% Triton X-100 in PBS and blocked for 30 min at 4˚C with 3% bovine serum albumin (Thermo Fisher Scientific, Inc.). The cells were then incubated overnight at 4˚C with anti-LC3 primary antibodies (1:400 dilution; SAB4300571, Sigma-Aldrich; Merck KGaA) in blocking buffer. After washing with PBS, the cells were incubated with Alexa Fluor 555 fluorescent secondary antibody (1:100; cat. no. bs-0295G-AF555; BIOSS) for 60 min at room temperature. The nuclei were then stained with 4',6-diamidino-2-phenylindole at 1:5,000 dilution for 5 min at room temperature. The cells were washed twice with PBS and fluorescence images were captured using a confocal microscope (Smartproof 5; Carl Zeiss AG). The intensity of staining was determined by measuring the integrated optical density (IOD) in 10 different fields for each sample.
3
Characterization of Printed Flexible Electrodes
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The photo image of the freshly printed electrode before and after the sintering curing was performed on an upright microscope (Olympus BX51). The geometrical parameters including the thickness and width after printing were tested on the surface profile D500 (KLA). The conductivity of the printed electrodes was tested by the four-probe method on a multimeter (DAQ6510, Keithley). JEOL 7600 and C-AFM (Asylum Research Cypher S) were used to test the surface conductivity and SEM images, respectively. The stretching test of printed VegPU/Ag electrodes (2 mm × 2 cm) was performed on a motorized force test stand (ESM303, Mark-10) with a speed of 2 mm min−1 and the corresponding resistance change was tested by a multimeter. The cycling stretching speed is 60 mm min−1 and the stretching rate dependence was tested with speeds from 10 to 1100 mm min−1. The confocal images were tested on a widefield confocal microscope (Smartproof 5, Zeiss). The biodegradation of VegPU was done by immersing a piece of film in the lipase enzyme solution (1 mg mL−1 in 0.01 M PBS buffer with pH 7.4).
4
Immunofluorescence Imaging of Growth Plate Chondrocytes
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Growth plate chondrocytes cultured in Confocal Dish were rinsed with PBS and fixed with 4% paraformaldehyde fixative for 15 min. Then they were permeated with 0.5% Triton X-100 at room temperature for 15 min. After washing with PBS, normal goat serum was dropped and sealed at room temperature for 30 min. Discard the sealing liquid, add the primary antibody directly according to the dilution ratio suggested in the primary antibody manual, and place it in a wet box at 4°C overnight. The next day, TBST was used for a full washing and fluorescence secondary antibody was added, and the cells were incubated at room temperature for 1 h without light. After that, PBST was used for washing, and DAPI was dropped and incubated for 5 min to avoid light. PBST was used for washing again, and images were observed and collected under a confocal microscope (Smart Proof 5, Carl Zeiss, Germany). The fluorescence intensity was determined by measuring the integrated optical density (IOD) in 10 different fields of each sample.
5
Cellular Protein Expression Analysis
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To determine the protein expression in cells, we used cellular IF. Specifically, after giving different treatments, we fixed the cells with 4% paraformaldehyde for 15 min, then permeabilized the cellular membrane with 0.3% Triton X-100 for 15 min, and washed with PBS 3 times. Then, the cells were blocked with 3% BSA for 1 h at room temperature. Incubation with primary antibody was performed overnight at 4 °C. On the next day, after washed with PBS 3 times, the cells were incubated with secondary antibody and DAPI at room temperature. The final image was taken with a confocal microscope (Smartproof 5, Carl Zeiss, Germany). The staining intensity was determined by measuring the integrated optical density (IOD) in 5 different fields for each sample. The fluorescence IOD value was obtained by calculating the total reaction intensity of all selected objects (target proteins) in a specific field of vision by Image J software.
6
Exosomal Uptake Experiment Protocol
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To perform exosomal uptake experiments, BMSC-Exos were labeled with DiI and then washed with PBS at 110,000 ×g for 70 min at 4 °C. Then, BMSC-Exos stained with DiI were added to macrophages for 6 h at a concentration of 50 µg/ml. After 6 h, the cells were washed with PBS, and the nuclei were stained with DAPI. To exclude nonspecific staining, when collecting exosomes for the last time, we collected the same volume of supernatant and performed DiI staining as a sham control. Finally, images were taken using a confocal microscope (Smartproof 5, Carl Zeiss, Germany).
7
Immunofluorescence Staining of β-catenin
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Cells (15 mm confocal dishes, 6 × 104 cells per well) were treated with DY, RZ or ZG for 24 h, which were then washed once with PBS solution. The cells were fixed with 4% paraformaldehyde for 30 minutes. Then the cells were washed in PBS solution and permeabilized with 0.1% Triton X-100. Blocked in 5% skim milk solution (30 min), the cells were blotted with anti-β-catenin and anti-β-actin for 2 h, after cells incubating with Alexa Fluor 594 AffiniPure Goat Anti-Rabbit IgG (H + L) for 1 h, they were washed with PBS and then mounted with DAPI Fluoromount-G™. Finally, the cells were observed and imaged by the confocal microscopy (Zeiss, Smartproof 5, Germany).
8
Laser-Induced Surface Morphology Analysis
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The surface morphology
of the laser-textured samples was studied by means of a scanning electron
microscope (Gemini SEM 500, ZEISS, German). The roughness of the sample
surface was analyzed using a laser scanning confocal microscope (LSCM,
Smartproof 5, ZEISS). The water contact angle (WCA) and the underwater
bubble CA of the rough aluminum sheet surface induced by the pulse
fiber laser were measured using an optical CA meter (KSV CAM 200).
To measure the WCA, a 4.0 μL drop of deionized water droplet
was generated and deposited on the filter surface (in air) using a
micrometric syringe by the sessile drop technique. To measure the
CA of underwater bubbles, the sample was carefully immersed inside
water and a 3.0 μL volume of air bubble was deposited on the
sample surface. The final CA value was averaged by measuring three
different points on the same surface at ambient temperature. The dynamic
process of water droplets and bubbles on the sample surface was captured
by IDS Imaging Systems (IDS, Germany) and Macro zoom lens (Nikon 60
mmf/2.8 d).
of the laser-textured samples was studied by means of a scanning electron
microscope (Gemini SEM 500, ZEISS, German). The roughness of the sample
surface was analyzed using a laser scanning confocal microscope (LSCM,
Smartproof 5, ZEISS). The water contact angle (WCA) and the underwater
bubble CA of the rough aluminum sheet surface induced by the pulse
fiber laser were measured using an optical CA meter (KSV CAM 200).
To measure the WCA, a 4.0 μL drop of deionized water droplet
was generated and deposited on the filter surface (in air) using a
micrometric syringe by the sessile drop technique. To measure the
CA of underwater bubbles, the sample was carefully immersed inside
water and a 3.0 μL volume of air bubble was deposited on the
sample surface. The final CA value was averaged by measuring three
different points on the same surface at ambient temperature. The dynamic
process of water droplets and bubbles on the sample surface was captured
by IDS Imaging Systems (IDS, Germany) and Macro zoom lens (Nikon 60
mmf/2.8 d).
9
Confocal Microscopy Analysis of Surface Erosion
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The surface profile of each specimen was measured before and after the erosion-abrasion cycle with widefield confocal microscopy (SmartProof 5 and ZEN Smartproof HF2 1.0; Zeiss, Oberkochen, Germany). Oriented on the reference markers an 1125 × 4500 µm2 image was recorded with a 10× magnification (C Epiplan-Apochromat 10×/0.4 objective lens, Zeiss, Oberkochen, Germany). The used settings were a fast scan with 150–200 z-levels in mode HDR and low resolution.
Image processing and evaluation was performed with a surface-metrology software (ConfoMap 7.4.8076; Zeiss, Oberkochen, Germany). Before the evaluation the underground was leveled over rotation using a least-square method, furthermore background noise was reduced by removing outliers. Oriented on the reference markers, the surface profile was determined before and after the erosion-abrasion cycle on a specific position of the specimens. Over a superimposition of the both surface profiles, the surface loss was determined from the average height over 0.5 mm in the middle of the specimen. For each sample, a three-fold determination was carried out.
Image processing and evaluation was performed with a surface-metrology software (ConfoMap 7.4.8076; Zeiss, Oberkochen, Germany). Before the evaluation the underground was leveled over rotation using a least-square method, furthermore background noise was reduced by removing outliers. Oriented on the reference markers, the surface profile was determined before and after the erosion-abrasion cycle on a specific position of the specimens. Over a superimposition of the both surface profiles, the surface loss was determined from the average height over 0.5 mm in the middle of the specimen. For each sample, a three-fold determination was carried out.
10
Surface Analysis of Enamel and Dentin
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Surface analysis was performed by two experienced operators (SB, BR) using widefield confocal microscopy (SmartProof 5, Zeiss, Germany) and ZEN Smartproof HF2 1.0 (Zeiss, Germany) for image acquisition. Of each enamel or dentin specimen, an 1125 × 4500 μm2 image with 10x magnification (C Epiplan-Apochromat 10×/0.4 objective lens) was taken before and after the experiment, and the surface profile was determined. Image acquisition was performed with a fast scan (150–200 z-levels) in low resolution in HDR mode. For image processing and evaluation, ConfoMap 7.4.8076 (Zeiss, Germany) was used. Further analyses of each image were performed, after the underground was leveled with a least-square method using rotation. The background noise was minimised by removing outliers. A three-fold determination of the surface loss of each specimen was done after superimposition of the surface profiles before and after the experiment. Surface loss was determined over a length of 0.5 mm in the middle of the specimen.
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