PDMC1-DMC1-mCherry or PFAS1-FAS1-mCherry were grown on V8 (pH7.0) plates for 5 days at room temperature in dark to form visible colonies. For probing DMC1 expression in different hyphal subpopulations, 50 µg/ml calcofluor white (stain cell wall) was added into the medium to enable the visualization of all fungal cell types. For sample preparation, colonies were embedded in low-gelling agarose (sigma-Aldrich) on the plates [59] (link). Immediately after gelling, agarose-embedded colonies were cut vertically in the middle and transferred to the cover glass with the longitudinal section facing the cover glass for the side view of the colonies. Cover the sample with 1∶9 PBS-Glycerol. An Olympus Fv1000 confocal laser scanning microscope and an Olympus IX81 spinning disk confocal microscope were used for the image acquisition. For Olympus Fv1000 confocal laser scanning microscope, calcoflour white and mCherry were excited with 488 nm argon and 543 nm He-Ne laser lines respectively using a dry 40× objective. For Olympus IX81 spinning disk confocal microscope, calcoflour white and mCherry were excited with 405 nm argon and 561 nm laser lines respectively with dry 40× objective. The data analyses were carried out with MetaMorph Microscopy Automation & Image Analysis Software (Molecular Devices, PA).
Fv1000 confocal laser scanning microscope
Manufactured by Olympus
Sourced in Japan, United States, Germany, China, United Kingdom, Australia
The FV1000 is a confocal laser scanning microscope designed for high-resolution imaging. It uses a focused laser beam to scan and excite fluorescent samples, capturing detailed images with improved contrast and resolution compared to traditional wide-field microscopy. The FV1000 is capable of optical sectioning, allowing for the reconstruction of three-dimensional structures from a series of two-dimensional images.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (27 mentions)
Fluoview software, by Olympus (26 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (24 mentions)
Vectashield, by Vector Laboratories (13 mentions)
Triton x 100, by Merck Group (11 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (11 mentions)
Hoechst 33342, by Thermo Fisher Scientific (10 mentions)
Fluoview fv10 asw 3.1 viewer software, by Olympus (9 mentions)
Bovine serum albumin (bsa), by Merck Group (6 mentions)
Paraformaldehyde (pfa), by Merck Group (6 mentions)
Sytox green, by Thermo Fisher Scientific (6 mentions)
Hoechst 33342, by Merck Group (5 mentions)
Ov100 small animal imaging system, by Olympus (5 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (5 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (5 mentions)
Calcein am, by Thermo Fisher Scientific (4 mentions)
Rabbit anti iba1, by Fujifilm (4 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (4 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (4 mentions)
738 protocols using fv1000 confocal laser scanning microscope
1
Visualization of DMC1 and FAS1 Expression in Fungal Hyphae
2
Immunocytochemical Analysis of LPS-Stimulated Primary Macrophages
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Purified primary cultured PVM/Ms at passage 3 were seeded in a glass well dish, and treated with LPS at 1μg/ml for 48 hours. The PVM/Ms were fixed in 4% PFA (pH 7.2) for 15 min, permeabilized in 0.5% Triton X-100 in PBS-BSA for 3 min, incubated with 10% goat serum, and incubated overnight at 4°C with primary antibody for rat monoclonal F4/80 (eBioscience, San Diego, CA) diluted 1:50 in 1% PBS-bovine serum albumin. After 3 washes in PBS, the samples were incubated with secondary antibodies, Alexa Fluor 568 goat anti-rat IgG (H+L) (Life Technologies, Eugene, OR) or lectin Griffonia simplicifolia IB4 (GS-IB4) conjugated to Alexa Fluor 488 (Life Technologies, Eugene, OR) diluted in 1% PBS-bovine serum albumin at 1:100 for 1 hour at room temperature. The cells were washed in 2 ml PBS (3 times for 10 min) and imaged under an FV1000 Olympus laser-scanning confocal microscope.
Purified PCs transfected with pmOrange2-N1 Vector (Clontech Laboratories, Inc., Mountain View, CA) were seeded in the glass well dish, and treated with LPS at 1 μg/ml for 48 hours, and imaged under an FV1000 Olympus laser-scanning confocal microscope with a standard 559 nm laser excitation line.
Purified PCs transfected with pmOrange2-N1 Vector (Clontech Laboratories, Inc., Mountain View, CA) were seeded in the glass well dish, and treated with LPS at 1 μg/ml for 48 hours, and imaged under an FV1000 Olympus laser-scanning confocal microscope with a standard 559 nm laser excitation line.
3
Immunofluorescence Analysis of β-Catenin Localization
4
Immunofluorescence and ROS Detection in C2C12 Cells
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Differentiated C2C12 cells were washed with PBS, fixed with 4% paraformaldehyde in PBS for 10 minutes and then permeabilized with 0.2% Triton X‐100 for 10 minutes at room temperature. The cells were then blocked with 2% BSA in PBS and incubated with an MF‐20 anti‐MHC Alex Fluor 488 antibody (eBioscience; Thermo Fisher Scientific, Inc) overnight. Images of microtubes were taken using a FV1000 laser scanning confocal microscope (Olympus, Japan).
To measure the formation of intracellular reactive oxygen species (ROS) in CCCP‐treated cells, C2C12 myotubes were incubated in serum‐free media containing 10 µmol/L 2′,7′‐dichlorofluorescin‐diacetate (DCF‐DA) cell permeant reagent (Invitrogen) for 20 minutes, and the cells were subsequently washed 3 times with PBS. DCF fluorescence images were obtained using a FV1000 laser scanning confocal microscope (Olympus, Japan).
To measure the formation of intracellular reactive oxygen species (ROS) in CCCP‐treated cells, C2C12 myotubes were incubated in serum‐free media containing 10 µmol/L 2′,7′‐dichlorofluorescin‐diacetate (DCF‐DA) cell permeant reagent (Invitrogen) for 20 minutes, and the cells were subsequently washed 3 times with PBS. DCF fluorescence images were obtained using a FV1000 laser scanning confocal microscope (Olympus, Japan).
5
Immunofluorescence Analysis of β-Catenin Localization
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For ICC, 2–3 × 105 cells were seeded onto glass coverslips in 6-well plates. After treatments, cells were washed twice wish PBS and fixed with ice cold 70% Acetone/30% methanol for 10 min. Fixed cells were blocked with 10% normal goat serum (Vector Lab, Burlingame, CA, USA) in 0.05% TBS-T for 30 minutes followed by addition of primary antibodies (CK5, mouse NCL-L-CK5, Leica Biosystems; β-catenin, 9587, Cell Signaling Technologies, 1:200) for 2 h, secondary antibodies (A11029, A11037, Invitrogen, 1:200) for 1 h, and counterstained with DAPI. Cells were imaged using the Olympus BX40 fluorescent microscope or Olympus FV1000 laser scanning confocal microscope. Individual cells were scored for membrane β-catenin coverage of 0–25% (low), 25–75% (medium), or 75–100% (high) in a blinded manner. IHC was performed as previously described (71 (link)) using the antibodies described above and imaged using the Olympus FV1000 laser scanning confocal microscope.
6
Immunofluorescence Analysis of Mouse Inner Ear
7
Visualizing NPR3 in PDLSCs via Immunofluorescence
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Immunofluorescence staining was performed to visualize NPR3 in PDLSCs. Briefly, cells were fixed with 4% paraformaldehyde for 30 min, followed by treatment with 0.5% Triton X-100 (Sigma-Aldrich) and 2% bovine serum albumin (BSA; Sigma-Aldrich). Then, the cells were incubated with primary antibodies against NPR3 (1:100; Abcam, ab97389) over night. The secondary antibodies for NPR3 were Alexa Fluor 488 AffiniPure donkey anti-rabbit IgG (1:100; Yeasen Biotech Company, Shanghai, China). Finally, the cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Abcam, ab104139), and the fluorescence images were acquired using an Olympus FV1000 confocal laser scanning microscope (CLSM; Olympus, Tokyo, Japan) and analyzed using FV10-ASW4.2 software.
8
Immunohistochemical Analysis of Murine Tissues
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Frozen murine tissues were fixed in cold acetone and 4% PFA before 8 μm cryosections were cut using a cryostat. Cryosections were then placed onto slides coated with Vectabond. anti-PDGFR-alpha (goat, 1:100, R&D system, MN), anti-Fibronectin (FN1) (rabbit, 1:100, abcam, MA), anti-Collagen VI (COL6) (rabbit, 1:200, abcam, MA), anti-PLAU (rabbit, 1:100, abcam, MA), anti-PROCR (Rabbit, 1:50, Bioss antibodies, MA), anti-BMP7 (Rabbit, 1:100, AVIVA system biology, CA), anti-SEMA3F (Rabbit, 1:50, Bioss antibodies, MA), and anti-PCSK6 (Goat, 1:100, antibodies-online, GA) were the primary antibodies used. Primary and secondary antibodies were diluted in 4% skim milk/1x TBS/0.1% Triton-X114. Each slide was washed twice with 1x TBS and treated with Fluoroshield mounting medium with DAPI (Abcam, ab104139) after incubation with the secondary antibodies. An Olympus FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan) was used to capture the immunohistochemically stained images and these images were analyzed through Fluoview FV10-ASW 3.1 Viewer software (Olympus, Tokyo, Japan).
9
Immunofluorescence Microscopy of MEFs
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Formaldehyde (4%) was added to fix MEFs for 10 min and then washed with phosphate-buffered saline (PBS). To block nonspecific binding, the MEFs were incubated in a blocking solution included 10% FBS and 0.1% Triton X-100 in PBS for 15 min, followed by overnight incubation with the primary antibodies. After washing with PBS-T, the cells were treated for 90 min with the following secondary antibodies, Alexa Fluor 594-labeled goat anti-rabbit immunoglobulin G (IgG, A11037; Life Technologies, Thermo Fisher Scientific; Waltham, MA, USA), Alexa Fluor 594-labeled goat anti-mouse IgG (A11032; Life Technologies, Thermo Fisher Scientific), and Alexa Fluor 488-labeled goat anti-rabbit IgG (A11034; Life Technologies, Thermo Fisher Scientific), followed by processing using 4′,6′-diamino-2-phenylindole (DAPI), to detect cell nuclei (1:3000; 5 mg/mL stock in DMSO; Merck, Darmstadt, Germany). Cells were mounted on slides using ProLong Gold Antifade mounting medium (Molecular Probes, P36034; Thermo Fisher Scientific), and cell immunofluorescence was detected using an Olympus FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan).
10
Optimized Lens Fixation and Immunolabeling
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Lenses were dissected and immediately fixed as previously reported.25 (link) For α-SMA labeling, a protocol employed by Nowak and colleagues,26 (link) was adapted, and optimized to fix the lenses without dissection from the eye. To do this, a small incision was first made at the corneal limbus and posterior pole of the enucleated eye, before placing the whole eye in 1% w/v PFA for 24 hours. Fixed lenses were dissected from the eye, cryoprotected, sectioned, and labeled as previously described.25 (link) For α-SMA labeling, sections were washed, and then incubated with α-SMA antibodies (1:200; Sigma-Aldrich, Darmstadt, Germany) overnight at 4°C. Sections were then labeled with Alexa Fluor 488 (1:200; Life Technologies, Carlsbad, CA, USA), washed, and then incubated in 4',6-diamidino-2-phenylindole (DAPI; 1:1000) and wheat germ agglutinin conjugated to an Alexa Fluor 594 (WGA; 1:100). Sections were then mounted with VECTASHIELD HardSet aqueous mounting medium (Vector Laboratories, Burlingame, CA, USA), and imaged using the Olympus FV1000 confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan). Raw data were processed and merged if required using Adobe Photoshop CS6 version 13.0 (Adobe Systems Pty. Inc., San Francisco, CA, USA).
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