JAK2 copy number was quantified by comparing the ratio of JAK2 to CEN9 probe signals. Tumor cells were examined directly using an Olympus AX70 epifluorescence microscope equipped with narrow band pass filters. Each slide was initially scanned at low power to identify appropriate areas of tumor tissue with clearly defined nuclei. The 100× objective was then used to score signals in 40–50 non-overlapping tumor cell nuclei to determine the average number of JAK2 and centromere 9 copies per cell. The risk of sampling error due to tissue heterogeneity was minimized by scoring at least 5 tumor areas for each specimen. In two cases, fewer nuclei were interpretable and these counts were normalized to a value for 40 cells. The ratio of the JAK2 and centromere 9 copy number averages was used to determine the presence of JAK2 gene amplification. Specimens with a JAK2:centromere 9 ratio ≥2 were scored as positive for JAK2 gene amplification. Specimens were considered to have JAK2 copy number gain if they had an average of ≥ 3 JAK2 signals.
Ax70 epifluorescence microscope
Manufactured by Olympus
Sourced in Japan, United States
The Olympus AX70 is an epifluorescence microscope designed for high-performance fluorescence imaging. It features a stable and durable frame, providing a reliable platform for a wide range of applications. The AX70 utilizes an epifluorescence illumination system, allowing for efficient excitation and detection of fluorescent samples.
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Lab products found in correlation
Tcs sp2 spectral confocal laser microscope, by Leica Microsystems (3 mentions)
Laf suite, by Leica camera (3 mentions)
Imagej, by Leica camera (3 mentions)
Dp70 digital camera, by Olympus (3 mentions)
A11036, by Thermo Fisher Scientific (2 mentions)
Ab84355, by Abcam (2 mentions)
U cmad3 camera, by Olympus (2 mentions)
Spotrt digital camera, by Olympus (2 mentions)
Coreldraw 2019, by Corel (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
A11004, by Thermo Fisher Scientific (1 mentions)
A11034, by Thermo Fisher Scientific (1 mentions)
A11029, by Thermo Fisher Scientific (1 mentions)
Dp71 ccd camera, by Olympus (1 mentions)
Alexa568 phalloidin, by Thermo Fisher Scientific (1 mentions)
Alkaline comet assay, by Bio-Techne (1 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (1 mentions)
20 protocols using ax70 epifluorescence microscope
1
Quantification of JAK2 Copy Number
2
Immunofluorescence Imaging of DNA Damage Foci
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For immunolocalisation, HeLa cells were cultured on 170 μm-thick coverslips (Marienfeld, Lauda-Königshofen, Germany) 24 h before transfection. After etoposide treatment, cells were washed twice with PBS with 500 μM MgCl2 and 500 μM CaCl2 (PBS-S). For whole cell detection, cells were directly fixed with 4% PFA after PBS-S washes. To detect the chromatin-associated fraction and to observe DNA damage foci, cells were permeabilised before fixation as follows: after PBS-S washes, cells were washed once with CSK buffer (10 mM Pipes, pH 7.0, 100 mM NaCl, 300 mM sucrose and 3 mM MgCl2) and incubated 10 min with CSK buffer containing 0.5% Triton X-100, 0.3 mg/ml RNase A (ThermoFisher Scientific) and complete protease inhibitor (Roche). Cells were then washed with PBS-S, fixed with 4% PFA for 20 min and washed with PBS-S. Before staining, cells were blocked with PBS-S/0.1% Tween 20 (PBS-S-T) containing 5% BSA. Cells were incubated with primary antibodies (Table S4) in PBS-S-T/5% BSA and then washed with PBS-S-T and incubated with appropriate fluorescent dye-coupled secondary antibody. After washes in PBS-S-T, coverslips were mounted on glass slides using ProLong® Gold antifade reagent with DAPI (Life Technologies). Imaging was performed using an AX70 epi-fluorescence microscope (Olympus) equipped with a Photometrics CoolSNAP MYO CCD camera. Images were analysed using ImageJ software.
3
Centrosome Staining and Counting
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Cells were seeded on glass slides and transfected or transduced with designated plasmids. Cells were fixed with 4% buffered formalin for 10 min and permeabilized with PBS with 0.1% Triton X-100 for 10 min. After blocking with 10% goat serum in PBS-T (PBS with 0.5% Tween 20), cells were incubated overnight at 4 °C with primary antibody γ-tubulin (ab84355, Abcam, Cambridge, MA, USA) or CEP170 (72-413-1, Thermo Fisher Scientific, Waltham, MA, USA) for staining, followed by secondary antibody incubation (Alexa Fluor 488- or 568-conjugated goat anti-rabbit (A11034 or A11036) or anti-mouse (A11029 or A11004, 1:1000, Invitrogen) for 1 h at room temperature. Stained cells were mounted in aqueous medium containing 4,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). Cells were analyzed by fluorescence imaging using an Olympus AX70 epifluorescence microscope equipped with a U-CMAD3 camera (Olympus, Tokyo, Japan). Centrosome counting was performed in a blinded manner by two observers.
4
Confluent HDF Monolayer Wound Healing
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Confluent HDF monolayers were scratched with a pipette tip. Cells were incubated in DPBS+, fixed at the indicated time points, stained with Alexa568-phalloidin (Molecular probes) and imaged with an AX70 epifluorescence microscope (Olympus) equipped with an Olympus DP71 CCD camera using a 4× objective. Images were analysed with ImageJ software by thresholding and automatically measuring the wound area. The migration area was calculated as the difference between the wound area at 0 h and 22 h post wounding.
5
Analysis of DNA Damage in CUL5-Deficient Cells
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For the analysis of DNA damage in CUL5-deficient cells, U-2 OS/centrin-GFP cells were transfected with siRNA duplexes against CUL5 mRNA (see above for protocol) for 72 h and efficiency of DNA repair was analyzed with or without 10 Gy IR using the alkaline Comet assay (Trevigen, Gaithersburg, MD, USA). Briefly, cells were trypsinized and pelleted by centrifugation. The cell pellet was then washed two times with PBS prepared without calcium or magnesium and cells were resuspended at a concentration of 1 × 105 cells/ml in PBS. Cells were then mixed with LMAgarose at a 1:10 ratio and 50 μl of this solution was pipetted onto Comet assay slides. The slides were placed at 4 °C in the dark for 10 min until the cell/agarose solution hardened. The slides were then immersed in cold lysis solution provided with the kit for 1 h at 4 °C in the dark. Slides were subsequently immersed in alkaline unwinding buffer (30 mM NaOH, 1 mM EDTA) for 1 h at 4 °C in the dark. Next, alkaline electrophoresis was performed using alkaline electrophoresis solution (300 mM NaOH, 1 mM EDTA) at 300 mA for 30 min. Slides were washed two times for 5 min each in dH2O and once for 5 min in 70% ethanol before being stained with SYBR Gold and analyzed on an Olympus AX70 epifluorescence microscope equipped with a SpotRT digital camera.
6
Immunofluorescence Imaging of DNA Repair Proteins
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Cells grown on 10 mm coverslips were permeabilized with 1% Triton-X-100 for 15 min, washed in phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde/PBS followed by blocking in 10% normal donkey serum (Jackson Immunoresearch, West Grove, PA, USA). Cells were incubated with primary antibody overnight followed by incubation with a Rhodamine Red- or Coumarin (AMCA)-conjugated secondary antibody (Jackson Immunoresearch, UK) for 2 h and mounted with 4′,6-diamidino-2-phenylindole (DAPI). Cells were analyzed using an Olympus AX70 epifluorescence microscope equipped with a SpotRT digital camera. Antibodies used were mouse anti-BRCA1, rabbit anti-CUL5 and rabbit anti-53BP1 obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). An anti-γH2AX antibody was obtained from Millipore. Mouse anti-Cep170 was a kind gift from Erich A. Nigg (Biozentrum, University of Basel, Switzerland)24 (link).
7
Immunofluorescence Staining of HPV E6 Protein
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The BIEC-c4 and HPV-BIEC cells were cultured in two separate T-75 flasks in DMEM/F12 medium supplemented with 5% FBS. After 48 h of incubation at 37 °C, cells were trypsinized with 0.05% trypsin–EDTA and cytospins with 1 × 105 cells were prepared using a cytofuge (Cytospin 3; Thermo Shandon Inc.). The cytospins were air dried at room temperature for 90 min, fixed in absolute acetone for 7 min, dried, stored at 4 °C overnight, and blocked with 1% chicken serum for 15 min. Immunofluorescence staining for HPV E6 protein was performed by incubating the HPV-BIECs cytospin for 90 min at room temperature with 150 µl of goat anti-HPV16E6 specific polyclonal IgG antibody (Santa Cruz Biotechnology, Dallas, TX, USA; sc-1584) at 1:50 dil. The slide was then washed 3 × with 1 × PBS and incubated at room temperature for 1 h with 200 µl of Alexa Fluor 488 conjugated chicken anti-goat IgG secondary antibody (Invitrogen, Grand Island, NY, USA; Cat. No. A-21467) at 1:200 dilution. Following incubation, the cytospin was washed with 1 × PBS three times and then counter-stained with 150 µl of 1.5 mM propidium iodide for 5 min. Then it was washed once, air dried, and mounted with permafluor mounting reagent. Photographs were taken at a 20 × magnification using an Olympus AX70 epifluorescence microscope (Olympus Corp., Tokyo, Japan).
8
Superoxide Detection in Cardiac Myocytes
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Cardiac myocytes were treated with 2.5 μM dihydroethidium (Invitrogen, Waltham, MA, USA, D23107) (30 min) and imaged by Olympus AX-70 epifluorescence microscope. Enhanced red fluorescence staining indicates enhanced the production of superoxide species.
9
Immunofluorescence Detection of HPV16 L1 Capsid
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Sf9 cells were infected with HPV recombinant baculovirus rBacV/HPV16 L1 on coverslips in 6-well plates. Three days later, the cells were fixed in 10% acetic acid, 50% ethanol, washed with phosphate-buffered saline (PBS) and then incubated with the mAbs of AE3 and AG7 for 1 h at 37°C followed by incubation with FITC-conjugated secondary antibody (1:80) (Invitrogen, USA). The fluorescence was detected under an Olympus AX70 epifluorescence microscope (Olympus, Tokyo, Japan).
10
Centrosome Visualization and Quantification
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