Histological and immunofluorescence microscopy analyses were performed as described previously27 (link). The resulting immune complexes were visualized with a goat anti-rabbit IgG H&L (Alexa Fluor 488) (ab150077, Abcam) or goat anti-mouse IgG H&L (Alexa Fluor 594) (ab150116, Abcam) secondary antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (VECTASHIELD Hardset Antifade Mounting Medium, H-1500, Vector Laboratories, Inc., Burlingame, CA, USA). Images were acquired using an upright fluorescence microscope (BX61-32FDIC, Olympus, Tokyo, Japan).
Bx61 32fdic
Manufactured by Olympus
Sourced in Japan
The BX61-32FDIC is a motorized upright microscope system designed for advanced research applications. It features a 32x digital zoom, motorized focus, and an integrated DIC (Differential Interference Contrast) module for enhanced contrast imaging of transparent specimens.
Automatically generated - may contain errors
Lab products found in correlation
Ab150077, by Abcam (1 mentions)
Ab150116, by Abcam (1 mentions)
Metamorph image analysis program, by Molecular Devices (1 mentions)
α tubulin, by Cell Signaling Technology (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
P nf κb, by Cell Signaling Technology (1 mentions)
Ds ri1 camera, by Nikon (1 mentions)
Goat anti rabbit igg h l alexa fluor 488 ab150077, by Abcam (1 mentions)
Eclipse ni u microscope, by Nikon (1 mentions)
8 protocols using bx61 32fdic
1
Fluorescence Microscopy of Immune Complexes
2
Microscopic Analysis of Luteolin-Induced Apoptosis
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Cell morphology was observed under an inverted phase-contrast microscope. Apoptotic changes were detected by Hoechst staining. Cells were seeded on cover slips and treated with luteolin for 24 h. The cover slips were then washed with PBS, and the cells were fixed and permeabilized with 100 % acetone at room temperature for 10 min. After washing with PBS, the cells were stained with Hoechst staining solution at 37 °C for 20 min. The cover slips were then washed with PBS, dried completely, and mounted on microscope slides. The slides were then observed under a fluorescence microscope (BX61-32FDIC; Olympus, Tokyo, Japan).
3
Papiliocin Inhibits LPS-Induced NF-κB Activation
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The 5 × 105 RAW 264.7 cells were seeded on the coverslips in 6-well plates. After 16 h, cells were pretreated with 10 μM papiliocin for 1 h before 50 ng/mL LPS treatment. After 30 min, the cells were fixed by 4% paraformaldehyde and permeabilized by 0.3% Triton X-100. After blocking (5% BSA), primary antibodies against p-NF-κB (Cell Signaling Technology, No. 3031, 1:50) or α-tubulin (Cell Signaling Technology, No. 3873, 1:200) were added for 90 min and then incubated with Alexa Fluor 488 (Invitrogen, No. A-28175) or Alexa Fluor 546 (Invitrogen, No. A-10040, 1:200) conjugated secondary antibodies for 40 min. We counterstained the nuclear region using Hoechst 33258 (Invitrogen, No. H3569, 1:200) and visualized by fluorescence microscope (BX61-32FDIC, Olympus) and analyzed by the MetaMorph Image analysis program (Molecular Devices).
4
Immunofluorescence Imaging of Tubulin
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Immunofluorescence assays were performed as previously described (32 (link)). Briefly, cells seeded onto glass coverslips and incubated overnight at 37°C in a 5% CO2 incubator, followed by treatment with STK899704. After 24 h, the cells were permeabilized, fixed with 100% acetone, and blocked with 0.1% (w/v) bovine serum albumin. The cells were incubated overnight with antibodies against α-tubulin at room temperature. Following incubation, the cells were treated with secondary anti-mouse antibodies for 1 h at room temperature. Fluorescence images were obtained using a BX61-32FDIC (Olympus, Tokyo, Japan) upright fluorescence microscope.
5
Immunofluorescence-based Protein Localization
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Immunofluorescence facilitates the identification of target protein localization. Cells were seeded on cover slips, fixed with acetone after 24 h and then washed with phosphate-buffered saline (PBS). The cover slips were incubated in PBS containing 0.5% respective primary antibodies specific to MUC1-C and β-catenin (1:500 with PBS) overnight at 4 °C. Fluorescein isothiocyanate-tagged secondary antibodies were then attached to primary antibodies for 1 h at room temperature. Nuclei were stained by DAPI (4,6-diamidino-2-phenylindole) staining. The cover slips were mounted on glass slides, and the cells were imaged by fluorescence microscopy (BX61-32FDIC, Olympus, Tokyo, Japan).
6
Histological Analysis of Muscle Tissue
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Paraffin-embedded tissue sections (5 μm) were cut in the cross-sectional plane at the mid-portion of the muscle, deparaffinized in xylene, and rehydrated through an ethanol/water series. Tissue slides were then subjected to H&E staining. All slides were evaluated under an Eclipse Ni-U microscope (Nikon, Tokyo, Japan), and images were acquired with a DS-Ri1 camera (Nikon) and analyzed using NIS Elements F4.00.00 version 4.0 (Nikon). For immunofluorescence microscopy, fixed frozen muscle sections (0.5 μm) were labeled with an anti-ferroportin antibody, and the resulting immune complexes were visualized with an Alexa Fluor 488 goat anti-rabbit IgG H&L (ab150077, Abcam) secondary antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (Vectashield Hardset Antifade Mounting Medium, H-1500, Vector Laboratories, Burlingame, CA, USA). Images were acquired using an upright fluorescence microscope (BX61-32FDIC, Olympus, Tokyo, Japan).
7
Zinc Accumulation Visualization in Seedlings
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Detection of Zn accumulation in roots of ZnONPs and Zn ion (8 and 12 mg L -1 ) treated seedlings was done with Zinpyr-1 treatment as described by Song et al. [33] after washing alternately with deionized water and in 10 mM ethylenediaminetetraacetic acid (EDTA). Images were taken using the fluorescent microscope and associated software (BX61-32 FDIC, Olympus, Japan).
8
Visualizing Stress Responses in Plant Roots
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To detect the superoxide generation, fresh roots were treated with fluorescent probe dihydroethidium (DHE) [13] . For detection of H 2 O 2 generation in roots, the fluorescent probe, 3′-(p-hydroxyphenyl) fluorescein (HPF) (Invitrogen, USA) was used [14] . The cell death in roots was determined using propidium iodide (PI) treatment with minor modifications from the method of Kwon et al. [15] . For the in vivo detection of lignification of roots, fresh roots were incubated in 1% (w/v) phloro-Acta Biologica Hungarica 67, 2016 glucinol-HCl solution for 5 min as described by Rogers et al. [25] . The DHE, HPF and PI treated roots were photographed with a fluorescence microscope and associated software (BX61-32 FDIC, Olympus, Japan). The phloroglucinol treated roots were photographed using a Nikon 80i (Nikon, Japan) microscope and related software.
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