The distribution of HSF1 was assessed by immunofluorescence as described previously [26 (link)]. Briefly, the cells were fixed with 4% paraformaldehyde (G1101-500ML, Servicebio, Wuhan, China) for 15 min, permeabilized with 1% Triton X-100 (G5060-100ML, Servicebio, Wuhan, China) for 10 min, and incubated overnight at 4 °C with anti-HSF1 primary antibody (A13765, 1:100, Abclonal, Wuhan, China). Next, the cells were incubated with Cy3-labeled goat anti-rabbit antibody (BA1032, Boster, Wuhan, China) for 1 h and DAPI (AR1177, Boster, Wuhan, China) for 15 min at room temperature. The distribution of HSF1 was then observed by fluorescence microscopy (IX73-A12FL/PH, OLYMPUS, Japan).
Ix73 a12fl ph
Manufactured by Olympus
Sourced in Japan
The IX73-A12FL/PH is an inverted fluorescence microscope designed for a wide range of cell and tissue imaging applications. It features a compact and modular design, allowing for flexible configuration and customization to meet the specific needs of your research. The IX73-A12FL/PH provides high-quality optical performance, enabling you to capture detailed images and videos of your samples.
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7 protocols using ix73 a12fl ph
1
Immunofluorescence-based Analysis of HSF1 Distribution
2
Morphological Characterization of MNs
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We used a stereomicroscope (JSZ6S, Jiangnan Novel Optics) to observe the morphology of MNs under the bright field and captured by a charge-coupled device camera (Oplenic digital camera). SEM images were characterized by a scanning electron microscope (HITACHI SU8010). The fluorescence photographs were observed by a fluorescence microscope (Olympus, IX73-A12FL/PH) with the pictures taken by charge-coupled device camera. The fluorescence images of the MNs were obtained with a laser scanning confocal microscope (Nikon, A1). The mechanical properties of MNs were tested with All-purpose Electronic Tester (Instron 5944). The thermograms were recorded by thermography (FLIR, E5xt).
3
Quantifying Myofibroblast Differentiation
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At 48 h after transfection, the level of α-smooth muscle actin (α-SMA) was measured using immunofluorescence assay. Briefly, after fixing in 4% formaldehyde (CAS#: 50-00-0, Sigma-Aldrich, USA) for 15 min and permeabilizing with 0.1% Triton™ X-100 (CAS#: 9002-93-1, Sigma-Aldrich, USA) for another 15 min at room temperature, cells were blocked with 5% normal goat serum (#5425, cell signaling technology) at room temperature for 1 h. Subsequently, cells were probed overnight at 4°C with α-SMA (1: 50, #34105, cell signaling technology), followed by incubation with fluorescein-conjugated secondary antibodies (1: 1000, #4409, Cell Signaling Technology) for 1 h at room temperature. Finally, after staining with DAPI (D9542, Sigma-Aldrich, USA) for 5 min, samples were imaged using a fluorescence microscope (IX73-A12FL/PH; Olympus, Japan) at 200× magnification.
4
Immunofluorescence Analysis of VSMCs
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Following treatment of VSMCs with PDGF and crocin, cells were fixed in 4% paraformaldehyde for 15 min and permeabilized in 0.1% Triton X-100 for another 15 min at room temperature. Subsequently, cells were blocked with 2% BSA for 30 min at room temperature followed by incubation with α-SMA antibody (1:400) at 4°C overnight. After washes with PBS for three times, cells were incubated with goat anti-rabbit IgG H&L Alexa Fluor® 488 (1:500) at room temperature for another 1 h. Finally, nuclei were stained with DAPI for 5 min. Images were taken under a fluorescence microscope (IX73-A12FL/PH; Olympus, Tokyo, Japan) at 200× magnification.
5
Chrysophanol Dose-Dependent Effects on HBL-52 Cells
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HBL-52 cells were grown in 6-well plates for 24 h and incubated with increasing doses of chrysophanol (0, 15, 30, 60, and 90 μM) for 48 h. Cells were counted using a inverted microscope (OLYMPUS, IX73-A12FL/PH). Cell colonies were assessed by selecting 10 views and calculating the average number of cells.
6
Quantifying Cellular Proliferation and Contractility
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The levels of α-smooth muscle actin (α-SMA) and Ki67 were detected by an immunofluorescence assay. Briefly, after being fixed in 4% formaldehyde for 15 min and permeabilized with 0.1% Triton™ X-100 for another 15 min, the cells were blocked in 5% goat serum for 1 h. Subsequently, cells were probed overnight at 4°C with α-SMA (#19245, cell signaling technology) or Ki67 (#12075, cell signaling technology), followed by incubation with secondary antibodies for 1 h. Finally, after being stained with DAPI (D9542, Sigma) for 5 min, the samples were imaged using a fluorescence microscope (IX73-A12FL/PH; Olympus, Japan).
7
Quantifying Neuronal Apoptosis in Cerebral Ischemia
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After behavioral evaluation, the rats were anesthetized and sacri ced by femoral artery blood collection, the cerebral tissue was quickly dissected and xed by 4% paraformaldehyde, para n embedding. sections of brain tissue were randomly selected. TUNEL method was applied to detect cells of apoptosis according to the instructions of TUNEL kit(Yeasen Biotech Co. Ltd., Shanghai, China). After TUNEL dyeing, anti uorescence quenching agent was added dropwise for sealing, neuronal apoptosis was observed by uorescence microscope (IX73-A12FL/PH, Olympus, Japan). Two sections of each sample were taken to acquire data from different ve elds of view in ischemic area at high power magni cation(20×), the numbers of TUNEL positive staining cells (green represents the positive cells) were analyzed by pathological image analysis system, and calculate the neuronal apoptosis rate [39] .
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