The worn surfaces of the composites were characterized using an optical microscope (U-25ND25, OLYMPUS, T2,Tokyo, Japan). Nanostructures of the tribofilms were imaged with a scanning electron microscope (SEM, 7610F, JEOL, Tokyo, Japan). Additionally, the changes in the chemical states of the tribofilms were analyzed with X-ray photoelectron spectroscopy (XPS, 250Xi, ESCALAB, Waltham, MA, USA). Moreover, slices of the tribofilm were prepared using focused ion beam (FIB) machining (Quanta 3D FEG, FEI, Hillsboro, OR, USA), and the fractured surfaces were then observed via high-resolution transmission electron microscopy (HR-TEM, TecnaiG2 TF20, FEI, Hillsboro, OR, USA).
U 25nd25
Manufactured by Olympus
Sourced in Japan
The U-25ND25 is a neutral density filter designed for use with Olympus microscopes. It reduces the intensity of light passing through the optical system, allowing for more precise control of illumination and improved image quality.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Roche (2 mentions)
Quanta 3d feg, by Thermo Fisher Scientific (1 mentions)
Tecnai g2 tf20, by Thermo Fisher Scientific (1 mentions)
Ab2185, by Abcam (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Model 260, by World Precision Instruments (1 mentions)
S120vc sensor, by Thorlabs (1 mentions)
U 25nd6, by Olympus (1 mentions)
A6625, by Merck Group (1 mentions)
6 protocols using u 25nd25
1
Characterization of Tribofilm Nanostructures
2
Quantifying Autophagic Induction via MDC
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The compound MDC, a specific autophago-lysosomal marker was used to analyze induction of autophagy [49 (link)–51 (link)]. For visualization of the autophagic vacuoles by fluorescence microscopy, cells were seeded on sterile cover slips and grown overnight. Following cytokine treatment, the cells were incubated for 10 min at 37°C with 0.05 mM MDC dissolved in PBS. The cover slips containing the cells were then washed with PBS and mounted with anti-fade mountant (containing DAPI). Intracellular MDC in the form of punctate dots were visualized using fluorescence microscopy. The images were taken at 100× under upright fluorescence microscope (Olympus, U-25ND25). For, fluorimetric measurement, cells after treatment were labeled with MDC for 10 min, washed with PBS and collected in 10 mM Tris-HCl (pH 8) containing 0.1% TritonX-100. Intracellular MDC was measured by fluorescence photometry (excitation 380 nm and emission 525 nm) in a microplate reader (Fluoroskan Ascent). An increase in MDC fluorescence upon treatment was expressed as fold change with respect to control.
3
Intracellular Nrf2 Localization by Immunofluorescence
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The intracellular distribution of Nrf2 was displayed by immunofluorescence technique. Paraffin-embedded tissue sections (4 µm) were dewaxed in xylene and rehydrated in graded ethanol solutions. The antigen retrieval method with citric acid solution was proceeded for 5 min at 95°C, and then was washed in PBS. Follwing that, the tissues were permeabilized with 0.5%Triton X-100 for 15 min and blocked in normal goat serum at room temperature for 30 min [28] (link). After incubation with the polyclonal Nrf2 primary antibody conjugated to FITC (1∶150, Biorbyt, UK) at 4°C overnight, the sections was washed triple with PBS. At last, the nuclei were counterstained with DAPI (Roche, Shanghai, China) and visualized using a fluorescent microscope (Olympus U-25ND25, Tokyo, Japan). The green staining was showed in cytoplasm and nucleus, while blue staining was in nucleus. The overlay color was considered to be positive. Ultimately, the results were evaluated by semi-quantitative analysis based on the proportion of the Nrf2 nucleoprotein to the number of nuclei in five fields of each slices at a 400 multiple signal magnification [29] (link).
4
Immunofluorescent Analysis of Lung Tissue
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Lung tissues were fixed in 4% paraformaldehyde for 48h, embedded in paraffin, and sliced into 5 μm sections based on routine protocols. Then, the sections were dewaxed in xylene, rehydrated with graded ethanol, and antigen retrieval by PH 9.0 EDTA. After rinsing in TBS three times for each 5min, slices were blocked with 10% donkey serum at 37 °C for 30min, and incubated with hydrated, and then incubated with rabbit polyclonal anti-HO-1/HMOX1 antibody (Proteintech, 10701-1-AP, 1:100 dilution) or anti- HIF-1a antibody (Abcam, ab2185, 1:100 dilution) at 4 °C overnight. Following washing triple in TBST, the sections were incubated with diluted fluorescent-labeled secondary antibodies at room temperature for 30min, and counterstained with 4’, 6-diamidino-2-phenylindole (DAPI) to indicate the nuclei for 10min in the dark. The images were captured using a fluorescent microscope (Olympus U-25ND25, Tokyo, Japan).
5
Intracellular Nrf2 Nucleoprotein Quantification
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To estimate the intracellular distribution of Nrf2 nucleoprotein, immunoflurescence analysis was perfomed as previously described [19 (link)]. Kidney tissues were embedded in paraffin, sectioned at 4μm, and then dewaxed in xylene and rehydrated with graded ethanol solutions. After protein blockade, the sections were incubated overnight at 4°Cwith the polyclonal Nrf2 primary antibody conjugated to FITC (dilution 1:150, Biorbyt, UK). Following washing triple in phosphate-buffered saline (PBS), slides were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI) (Roche, Shanghai, China) to visualize the nuclei. The images were captured by a fluorescent microscope (Olympus U-25ND25, Tokyo, Japan) coupled to a digitial camera. The overlay color of blue staining in nucleus, accompanied with green staining both in cytoplasm and nucleus was considered to be positive. A semiquantitative numeric scores were determined for kidney samples based on the percentage of positive protein in five fields of each slices at a 400 multiple signal magnification using Image-pro plus software (Image pro plus 6.0, media Cybernetics, USA) [32 (link), 33 (link)].
6
Optogenetic Neural Stimulation Protocol
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For iontophoresis of ACh, a sharp glass pipette ($15 MU) was filled with 10 mM acetylcholine chloride (A6625, Sigma) dissolved in external saline. ACh was ejected into the MB calyx by a brief (500 ms) positive current pulse using an iontophoresis unit (Model 260, World Precision Instruments).
Stimulation of optogenetic probes LED/mercury light stimulation Wide-field optical stimulation was achieved by high power LEDs (M470L2 and M590L3 for ReaChR and CsChrimson, respectively, Thorlabs) or filtered light from a mercury lamp (for Arch). An LED (M470L2) with peak output at wavelength of $470 nm was used for both the activation of ReaChR and excitation of Citrine tagged to ReaChR. Because ReaChR is sensitive to a broad spectrum of light (Lin et al., 2013) , blue light was sufficient to make the PNs expressing ReaChR fire at $200 Hz (Figure S3A). Light from an LED or a mercury lamp was collimated and delivered to an upright microscope (BX51Wl, Olympus) equipped with a 40x water-immersion objective lens (NA 0.80). LED light was pulsated at 80 Hz. Neutral-density filters (U-25ND25 or U-25ND6, Olympus) were used to stimulate the cells at lower intensities. All the reported optical intensity of LED light was measured at the back aperture of the objective lens (S120VC sensor, Thorlabs).
Stimulation of optogenetic probes LED/mercury light stimulation Wide-field optical stimulation was achieved by high power LEDs (M470L2 and M590L3 for ReaChR and CsChrimson, respectively, Thorlabs) or filtered light from a mercury lamp (for Arch). An LED (M470L2) with peak output at wavelength of $470 nm was used for both the activation of ReaChR and excitation of Citrine tagged to ReaChR. Because ReaChR is sensitive to a broad spectrum of light (Lin et al., 2013) , blue light was sufficient to make the PNs expressing ReaChR fire at $200 Hz (Figure S3A). Light from an LED or a mercury lamp was collimated and delivered to an upright microscope (BX51Wl, Olympus) equipped with a 40x water-immersion objective lens (NA 0.80). LED light was pulsated at 80 Hz. Neutral-density filters (U-25ND25 or U-25ND6, Olympus) were used to stimulate the cells at lower intensities. All the reported optical intensity of LED light was measured at the back aperture of the objective lens (S120VC sensor, Thorlabs).
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