For BiFC, the Avr1b ORF sequence was cloned into pSAT1-cEYFP-C1-B to generate the C-terminal in frame fusion with cEYFP (Forward primer: 5′-GACTAAGCTTCGATGCGTCTATCTTTTGTGC-3′; reverse primer: 5′-AGTCGGATCCTCACTGGTGGTGCTGGTGGTG-3'), whereas GmPUB1-1Δ71-397 was introduced into pSAT1-nEYFP-N1 to form the N-terminal in frame fusion with nEYFP (Forward primer: 5′-GACTAGATCTCGATGCAATCTTGGTGCACCCTC-3′; reverse primer: 5′-CAGGATCCCGGGTTCCTTTGCCCTCTCCTTAG-3′). Onion lamellas were placed inside Petri dishes containing moist filter papers and then bombarded with gold particles coated with each combination of plasmids: GmPUB1-1Δ71-397-YFPN/YFPC- Avr1b fusion plasmids or YFPN/YFPC as the negative control. Onion inner layer were bombarded at 1350 PSI Helium pressure and kept in dark at 22°C post-bombardment. The EGFP signal was detected 24 h after the bombardment with a fluorescence microscope (Carl Zeiss Axiostar Plus). The microscopic field was viewed under green isothiocyanate filters. Fluorescence was detected after 24 h with a fluorescence microscope (Carl Zeiss Axiostar Plus).
Axiostar plus
Manufactured by Zeiss
Sourced in Germany, United States, Italy
The Axiostar Plus is a versatile and reliable microscope from Zeiss, designed for a wide range of laboratory applications. It features high-quality optics and a robust construction, providing consistent performance and durability.
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Lab products found in correlation
Axiovision 4, by Zeiss (3 mentions)
Axiocam mrc5, by Zeiss (3 mentions)
Powershot g6, by Canon (2 mentions)
Paraformaldehyde (pfa), by Wuhan Servicebio Technology (2 mentions)
Axiocam erc5, by Zeiss (2 mentions)
Tunel fluorescein kit, by Roche (2 mentions)
Moticam 5, by Zeiss (2 mentions)
Kling on hier, by Biocare Medical (2 mentions)
Histostain plus, by Thermo Fisher Scientific (2 mentions)
Axiocam mrc, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Nanog, by Merck Group (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Acridine orange, by Merck Group (1 mentions)
Axio imager a2, by Zeiss (1 mentions)
Rm2165, by Leica (1 mentions)
Axiocam hrc, by Zeiss (1 mentions)
Camera lucida, by Zeiss (1 mentions)
Axiocam mrc5 camera, by Zeiss (1 mentions)
Trypsin, by Harvard Bioscience (1 mentions)
137 protocols using axiostar plus
1
Bimolecular Fluorescence Complementation Assay
2
Bladder Wall Histology Analysis
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Urinary bladder tissue samples were fixed in 10% formaldehyde for 24 h. Afterward, they were dehydrated, diaphanized, and embedded in paraffin. Two histological sections per animal, non-seriated of 5 μm thickness, were collected and stained in hematoxylin–eosin for thickness analysis of bladder wall layers in a light field microscope (Zeiss-Axiostar Plus) and picrosirius red for analysis of type I and type III collagen fibers by polarized light in the microscope (Zeiss-Axiostar Plus).
3
Analyzing Yeast Sporulation and Spore Viability
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Cells were collected from sporulation cultures, pelleted via centrifugation (1500 g, 2 min), and fixed in 80% (v/v) ethanol for a minimum of 2 hr at 4 °C. The cells were pelleted again (1500 g, 2 min) and resuspended in PBS with 1 µg/ml DAPI. The proportion of cells containing one or multiple nuclei were counted using a fluorescent inverted microscope.
Tetrad/ dyad counts S288C and A364A: WT and pho92Δ strains were patched on YPD plates and incubated for 2 days. The patches were transferred on SPO plate and further incubated for 1 week at room temperature. Colonies were suspended in 10 µL of water and observed under 40 x objective (Axiostar Plus Zeiss). Number of packaged spores per ascus were counted for 200 asci.
Spore viability SK1: Diploid cells were patched from YPD agar plates to SPO agar plates and incubated for 3 days at 30 °C or 37 °C. Subsequently at least 40 tetrads (160 spores) were dissected and incubated at 30 °C for 72 hr on YPD agar plates before spore survival was assessed.
Tetrad/ dyad counts S288C and A364A: WT and pho92Δ strains were patched on YPD plates and incubated for 2 days. The patches were transferred on SPO plate and further incubated for 1 week at room temperature. Colonies were suspended in 10 µL of water and observed under 40 x objective (Axiostar Plus Zeiss). Number of packaged spores per ascus were counted for 200 asci.
Spore viability SK1: Diploid cells were patched from YPD agar plates to SPO agar plates and incubated for 3 days at 30 °C or 37 °C. Subsequently at least 40 tetrads (160 spores) were dissected and incubated at 30 °C for 72 hr on YPD agar plates before spore survival was assessed.
4
Pluripotency Marker Immunofluorescence Assay
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The expression of pluripotency antigens was determined for each healthy volunteer. NC were stained for 2 hours with antibodies against SSEA-4 (clone MC-813-70, mouse monoclonal IgG), Tumor Rejection Antigen (TRA-1-81, clone TRA-1-81, mouse monoclonal IgM), OCT-4A/4B (clone 9E3.2, mouse monoclonal IgG), and NANOG (goat polyclonal) (Millipore, Molsheim, France), as previously described in detail [21 (link)]. Appropriate secondary FITC or tetramethylrhodamine-5-isothiocyanate (TRITC) goat anti-mouse IgG or IgM and FITC-goat anti-rabbit (Beckman Coulter, Marseille, France) were added for 1 h. The nuclei were labelled with 4′,6-Diamidino-2-phenylindole (DAPI) complemented with Vectashield (Vector Laboratories, Abcys, Paris, France). Cells stained with secondary antibodies only were used as negative controls. Slides with H9 and/or HUES3 hESC lines were stained similarly and used as positive controls. Fluorescence images were recorded with the AxioVision 4.7 “Full support” system attached to a fluorescent microscope Axiostar Plus Zeiss and captured by AxioCam ICC 1 R3 Cameras (Lordil, Villers-les-Nancy, France).
5
Micronuclei Quantification in Erythrocytes
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Drop blood samples were taken from the animals before they died (experimental Groups 1, 2, 3, and 5) or euthanized (Groups 4, 6, and 7). Two smears were made on cleaned microscope slides. The smears were air-dried, fixed in absolute ethanol for 10 minutes, and stained with acridine orange (CAS No. 10127023, Sigma-Aldrich).
The micronuclei in each sample were scored manually using a binocular microscope (Carl Zeiss, Axiostar Plus) with a fluorescent filter (IVFL, 450–490 nm). The number of micronucleated erythrocytes (MNE) in 10,000 total erythrocytes and the polychromatic erythrocytes (PCE) in 1000 total erythrocytes were evaluated.
The micronuclei in each sample were scored manually using a binocular microscope (Carl Zeiss, Axiostar Plus) with a fluorescent filter (IVFL, 450–490 nm). The number of micronucleated erythrocytes (MNE) in 10,000 total erythrocytes and the polychromatic erythrocytes (PCE) in 1000 total erythrocytes were evaluated.
6
Immunohistochemical Detection of HS Antigen
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For immunohistochemistry, 3- to 3.5-μm sections of formalin-fixed, paraffin-embedded tissue sections were deparaffinized and antigen was retrieved by treatment with unmasking solution at 95–98°C for 20 min. Immunostaining was performed using biotin-free polyvalent DAB system (Spring Bioscience, USA) according to the manufacturer’s instructions. Briefly, endogenous peroxidase was blocked by incubation with peroxidase inhibitor (30 min at 37°C), unspecific staining was blocked with 5% Fetal Bovine Serum in PBS (20 min at 37°C), and the primary anti-HS mouse monoclonal antibody (Abnova, USA) (1:200) and secondary anti-mouse HRP (1:2000) were used for immunostaining (1 h at 37°C). Staining patterns were visualized by incubation with DAB (15 min at 37°C), counterstained with hematoxylin–eosin, and observed by light microscopy (Axiostar Plus, Carl Zeiss).
7
Microscopic Examination of Prepared Samples
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The obtained preparations were examined at magnification modes 103×; 247× and 413×; 600×. Microscopes used include: Axiostar plus (Carl Zeiss, Oberkochen, Germany) with an image capturing device and monitor with lenses A-plan 10×/0.25, A plan 20×/0.45 ph 2, A plan 40×/0.65; light microscope Axio Imager A2 (Carl Zeiss, Oberkochen, Germany); and electronic microscope Merlin (Carl Zeiss, Oberkochen, Germany). Image analysis and morphometry were performed using Axio Vision 4.8.2 (Carl Zeiss, Oberkochen, Germany) and VideoTest Size 5.0 (Moscow, Russia).
8
Histological Assessment of Tissue
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Specimens were embedded in paraffin and subsequently sectioned using a microtome (RM2165- Leica Microsystem Inc.; Buffalo Grove, IL, USA). One hematoxylin-eosin (H&E) stained 5-μm section from each sample was used for the microscopic evaluation of inflammation intensity, collagen degradation, and pulp tissue organization. Microscopic analysis was performed under trinocular microscopy (Axiostar Plus- Carl Zeiss; Jena, Turingia, Germany).
9
Phlebotomine Sand Fly Collection and Identification
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Phlebotomines were collected at a forested site, approximately 300 km south of Itaituba (BR163 highway), Pará State, Brazil (06º19′41.3″S 55º47′31.5″W). This region belongs to the Tapajós Mineral Province in the middle Tapajós basin (Amazonian biome). The vegetation comprises closed and open ombrophilous rainforest situated in an area of dry-land. The substrate includes a thin litter layer resulting from logging. Phlebotomines were collected with ground-level (0.5 m) CDC light traps, Shannon traps, and aspiration at the foot of trees with a ‘Nasci’ mechanical aspirator.
One specimen was prepared and mounted in berlese media, according to Ryan (1986) , and the others were prepared and mounted in Canada balsam (Reagen), according to the technique adapted from Forattini (1973) . Measurements were obtained using the software AxioVision version 4.7, coupled with a previously calibrated microscope–camera–computer system (Zeiss Axiostar plus; camera AxioCam HRC, Germany). Specimens were drawn using a camera lucida (Zeiss, Germany). Nomenclature and terminology are followed as discussed in Galati et al. (2017) (link).
One specimen was prepared and mounted in berlese media, according to Ryan (1986) , and the others were prepared and mounted in Canada balsam (Reagen), according to the technique adapted from Forattini (1973) . Measurements were obtained using the software AxioVision version 4.7, coupled with a previously calibrated microscope–camera–computer system (Zeiss Axiostar plus; camera AxioCam HRC, Germany). Specimens were drawn using a camera lucida (Zeiss, Germany). Nomenclature and terminology are followed as discussed in Galati et al. (2017) (link).
10
Histological Sample Preparation Protocol
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Scaffolds for histology were prepared exactly the same way as for proliferation assay. Samples were then rinsed in phosphate-buffered saline (pH 7.4), incubated in 5% sucrose (48°C, 30 min), embedded in Tissue-Tek cryoprotective media (Sakura, Zoeterwoude, The Netherlands), and frozen in liquid nitrogen. Serial cryosections (10 µm) were cut, mounted on SuperFrost glass slides (Menzel-Gläser, Braunschweig, Germany), fixed in acetone, and dried at room temperature. Samples were stained with hematoxylin and eosin (H + E) using a standard protocol. Finally, samples were embedded and analysed by light microscopy (Axiostar Plus, Zeiss, Munich, Germany).
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