CPCFC was detected in vivo by western blot and immunohistochemical analysis as previously described [6 (link), 7 ]. For western blots, 10 flies were extracted in lysis buffer (50 mM Tris–HCl pH 7.5, 8 M urea, 0.2 M NaCl, 0.1% SDS) and using the BCA Protein Assay Kit (Beyotime Biotechnology) to quantify extracted protein. The polyclonal antibody of CPCFC and α-tubulin were respectively generated in rabbits with the synthetic recombinant peptide (AtaGenix) (Additional File 3 : Table S3). The CPCFC and α-tubulin antibody were diluted by antibody diluent (Beyotime Biotechnology) at 1:1,000. For immunohistochemical analysis, 8 μm sections of the pronotum were prepared as described above, and imaged by LSCM. The CPCFC antibody was diluted by PBST (phosphate buffered saline pH7.5) with 1:200. The fluorescence intensity was analyzed using ZEN v2.3 (blue edition) software (Carl Zeiss).
Zen v2.3 blue edition software
Manufactured by Zeiss
ZEN v2.3 blue edition is a comprehensive microscope imaging software developed by Zeiss. It provides a user-friendly interface and advanced functionality for acquiring, processing, and analyzing microscopic images. The software supports a wide range of Zeiss microscopes and imaging systems.
Automatically generated - may contain errors
Lab products found in correlation
Imager m2, by Zeiss (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Antibody diluent, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Lsm 880 airyscan microscope, by Zeiss (1 mentions)
Inkredible, by Cellink (1 mentions)
Stemi 508 stereo microscope, by Zeiss (1 mentions)
Axiocam 105 mono, by Zeiss (1 mentions)
Axio imager m2, by Zeiss (1 mentions)
Live dead kit, by Thermo Fisher Scientific (1 mentions)
Lsm 880 airyscan, by Zeiss (1 mentions)
Axio imager 2, by Zeiss (1 mentions)
9 protocols using zen v2.3 blue edition software
1
Detection and Quantification of CPCFC Protein
2
Imaging of LGTV-infected Salivary Glands
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Dissection followed by mock and LGTVGFP infection of SGs, performed as described above, was completed. SGs were then collected at 180 hpi and fixed for a minimum of 24 h in 4% paraformaldehyde at 4°C. Individual SGs were washed briefly in 1× PBS following fixation and mounted on microscope slides using ProLong Gold Antifade reagent (Life Technologies) and 12-mm-diameter coverslips (VWR). Mounts were set for at least 24 h at 4°C prior to being imaged on a LSM710 confocal microscope (Zeiss). Images were acquired using a frame size of 2,048 by 2,048 pixels and 1.0× digital zoom with an EC Plan-Neofluar 10×/0.30 numerical aperture (N.A.) or a Plan-Apochromat ×63/1.40 N.A. oil objective. Z stacks used a stack thickness of 1 µm.
Images were observed using Zeiss ZEN v2.3 (blue edition) software. For both mock- and LGTV-infected SGs, ×10 and ×63 images were observed using a software display setting for both red and green channels at settings of 25 and Auto Best Fit, respectively. Z stack images (×10 and ×63) were observed with the software display setting of Auto Best Fit. Movies of the processed Z stack images were recorded with Imaris v8.4.1 software using a gamma correction of 2.0 with both red and green channels at 400 frames.
Images were observed using Zeiss ZEN v2.3 (blue edition) software. For both mock- and LGTV-infected SGs, ×10 and ×63 images were observed using a software display setting for both red and green channels at settings of 25 and Auto Best Fit, respectively. Z stack images (×10 and ×63) were observed with the software display setting of Auto Best Fit. Movies of the processed Z stack images were recorded with Imaris v8.4.1 software using a gamma correction of 2.0 with both red and green channels at 400 frames.
3
3D Bioprinting of GelMA-Nanoliposomes
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GelMA embedded, DiO-loaded nanoliposomes bioink (containing Irgacure 2959 photoinitiator, 0.1% in PBS pH = 7.4) was used to 3D bioprint disc-shaped constructs using a pneumatic extrusion bioprinter INKREDIBLE+ (CELLINK, Gothenburg, Sweden) equipped with a 23G nozzle, operating at pressures ranging from 60–70 kPa. GelMA-nanoliposomes were UV crosslinked (360–480 nm) for 40 s to generate nanocomposite hydrogels (Omnicure S-2000, 0.86 W/cm2). Confocal laser scanning microscopy imaging was performed in an LSM 880 Airyscan microscope (Carl Zeiss, Oberkochen, Germany) equipped with GaAsP/PMT detectors and a 20x/NA 0.8 Plan-Apochromat objective. Acquired data was post-processed in Zeiss ZEN v2.3 blue edition software.
4
OPN Expression in PLMA Hydrogels
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The expression of OPN on the PLMA hydrogels was assessed after 21 d of culture by fluorescence imaging. After wash with PBS, the hydrogels were fixed with 10% (v/v) formalin for 2 h. The samples were then permeabilized with 1% (v/v) Triton X-100 in PBS, blocked with 3% (w/v) BSA during 1 h and incubated overnight with mouse anti-human OPN antibody (1:100 in 5% (v/v) FBS/dPBS) at 4 °C. Cells were then rinsed in PBS and incubated with Alexa Fluor 488 goat anti-mouse (1:400 in 5% (v/v) FBS/dPBS) for 1 h at RT in the dark. For cell nucleus staining, the samples were incubated for 5 min with DAPI (1:1000 in PBS, original solution at 5 mg mL−1). After washing with PBS, hydrogels were examined using an upright fluorescence microscope (Zeiss Imager M2) equipped with a monochromatic digital camera (AxioCam MRm, 3Mpix). Image processing was performed by using the ZEN v2.3 blue edition software (Carl Zeiss Microscopy GmbH).
5
Quantifying In Vitro Biomineralization
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In vitro biomineralization was assessed at 21 d of culture either by hydroxyapatite crystals analysis or Alizarin Red S staining. In order to detect hydroxyapatite crystal, OsteoImage™ Mineralization Assay kit was used according to the manufacturer's instructions. After fixation with 10% (v/v) formalin, the samples were incubated with Osteoimage™ Staining reagent (1:100 in PBS) for 30 min. Afterward, the samples were incubated for 5 min with DAPI (1:1000 in PBS, original solution at 5 mg mL−1). After washing with PBS, hydrogels were examined using an upright fluorescence microscope (Zeiss Imager M2) equipped with a monochromatic digital camera (AxioCam MRm, 3Mpix). Image processing was performed by using the ZEN v2.3 blue edition software (Carl Zeiss Microscopy GmbH). For Alizarin Red S mineralization assay, hydrogels were fixed and washed as previously mentioned. After, the samples were incubated with 1 mL of Alizarin Red S (4 × 10−4 M, pH 4.2) for 1 h at RT. The staining solution was then removed, and the cells rinsed three times with PBS. The images were acquired using a Stemi 508 Stereo Microscope (Zeiss).
6
Photopolymerizable Hydrogel Nanocomposites
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PLMA were synthesized using a previously reported procedure [9 (link)]. Briefly, human PL was chemically modified by reaction with MA. The excess MA was removed by dialysis against deionized water, and the PLMA freeze-dried and kept at 4 °C until further use. Lyophilized PLMA was dissolved in a solution of 0.5% (w/v) Irgacure 2959 in PBS to a final concentration of 15% (w/v) PLMA. Afterward, MSNCaPDex was mixed in 15% (w/v) PLMA solution to a final concentration of 1% (w/v) nanoparticles. PLMA hydrogel nanocomposite was prepared by transferring the solution to PDMS molds with 6 mm diameter followed by ultraviolet (UV) irradiation (95 mW cm−2).
To assess MSNCaPDex dispersion in the PLMA hydrogel composites, a fluorescent molecule (PDI) was incorporated in the nanoparticles structure (PDI-MSNCaPDex), as previously reported [25 (link)]. PLMA hydrogel nanocomposites with PDI-MSNCaPDEx were imaged in an upright fluorescence microscope (Zeiss Imager M2) equipped with a monochromatic digital camera (AxioCam MRm, 3Mpix). Image processing was performed with the ZEN v2.3 blue edition software (Carl Zeiss Microscopy GmbH).
To assess MSNCaPDex dispersion in the PLMA hydrogel composites, a fluorescent molecule (PDI) was incorporated in the nanoparticles structure (PDI-MSNCaPDex), as previously reported [25 (link)]. PLMA hydrogel nanocomposites with PDI-MSNCaPDEx were imaged in an upright fluorescence microscope (Zeiss Imager M2) equipped with a monochromatic digital camera (AxioCam MRm, 3Mpix). Image processing was performed with the ZEN v2.3 blue edition software (Carl Zeiss Microscopy GmbH).
7
Live/Dead Hydrogel Assay
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At predetermined time points, PLMA hydrogels were incubated in a solution of 2 μL of calcein AM 4 × 10−3 M solution in DMSO and 1 μL of propidium iodide 1 mg mL−1 in 1,000 μL of PBS at 37 °C during 30 min. After washing with PBS, hydrogels were examined using an upright fluorescence microscope (Zeiss Imager M2) equipped with a monochromatic digital camera (AxioCam MRm, 3Mpix). Image processing was performed by using the ZEN v2.3 blue edition software (Carl Zeiss Microscopy GmbH).
8
Fluorescence Microscopy of Cell Cultures
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Fluorescence microscopy micrographs of the cells cultured with the medium containing the extracts released from the (HTCC/ ALG) 200 , (HTCC/ALG/HTCC/FITC-BSA) 100 and (HTCC/ALG) 200 / HTCC/FITC-BSA FS membranes were acquired in an upright motorized widefield fluorescence microscope (Axio Imager M2, Carl Zeiss, Jena, Germany) equipped with a 200 W HXP lamp, a 3.0 Mpix monochromatic camera (Axiocam 105 mono; Carl Zeiss, Jena, Germany), and a 5x objective (Carl Zeiss, Jena, Germany). Acquired data were processed in the Zeiss ZEN v2.3 blue edition software.
9
Viability Imaging of Cell-Laden Hydrogels
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To evaluate cell viability, cell-laden alginate filaments and cross-sections of cell-laden XG-GMA hydrogels were incubated in Calcein-AM/Propidium iodide (PI) (Live/Dead kit, ThermoFisher Scientific) for 20 min, according to the manufacturer's protocol. Widefield (Zeiss, Axio imager 2) and confocal laser scanning (Zeiss, LSM 880 Airy Scan) microscope systems were used to image the stained cell-laden structures. Acquired data was processed in Zeiss ZEN v2.3 blue edition software.
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