Fluorescent images of skeletal metastases were acquired using a Zeiss AX70 microscope (Carl Zeiss, Oberkochen, Germany) connected to a Nuance Multispectral Imaging System (CRI, Guelph, Ontario). Digital images were analyzed and processed with the Nuance Software (v. 2.4). Microscope and software calibration for size measurement was performed using a TS-M2 stage micrometer (Oplenic Optronics, Hangzhou, China).
Ax70 microscope
Manufactured by Zeiss
Sourced in Germany
The AX70 microscope is an optical microscope designed and manufactured by Zeiss. It features advanced optics and illumination systems to provide high-quality images for a variety of applications. The AX70 microscope is capable of magnifying specimens up to a certain degree, but the specific magnification capabilities are not included in this factual description.
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Lab products found in correlation
Anti mouse conjugated to alexa fluor 546, by Thermo Fisher Scientific (2 mentions)
Anti rabbit conjugated to alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Zen 2.3 lite software, by Zeiss (2 mentions)
D luciferin, by PerkinElmer (1 mentions)
Ivis lumina xr, by PerkinElmer (1 mentions)
24 well plate, by Sarstedt (1 mentions)
Alexa fluor 488, by Merck Group (1 mentions)
Zen software, by Zeiss (1 mentions)
Alexa fluor 647, by Merck Group (1 mentions)
Anti connexin 43, by Merck Group (1 mentions)
Dapi fluoromount g mounting medium, by Southern Biotech (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Serva Electrophoresis (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Apotome microscope, by Zeiss (1 mentions)
β tubulin, by Abcam (1 mentions)
6 protocols using ax70 microscope
1
Skeletal Metastasis Imaging Analysis
2
In Vivo Bioluminescence Imaging of Tumors
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Continuous long-term in vivo monitoring of bioluminescence was performed using the IVIS Lumina XR (PerkinElmer, Waltham, MA). Mice were administered weekly via i.p. with D-Luciferin, K + Salt Bioluminescent Substrate (PerkinElmer, Waltham, MA) and analysis was completed using Caliper Life Science's Living Image software. Animals were sacrificed and tissues were fixed, decalcified in 0.5 M EDTA if necessary and frozen in O.C.T. embedding medium (Electron Microscopy Sciences, Hatfield, PA, USA). Serial tissue sections of 80 μm in thickness were obtained using a Microm HM550 cryostat (Mikron, San Marcos, CA). Sections of each soft-tissue organs and hind legs were transferred on glass slides, stored at −20°C and examined for cancer cells using a Zeiss AX70 microscope (Carl Zeiss, Oberkochen, Germany) connected to a Nuance Multispectral Imaging System (CRI, Guelph, ON). Digital images were analyzed and processed with the Nuance Software (v. 2.4). Microscope and software calibration for size measurement was regularly performed using a TS-M2 stage micrometer (Oplenic Optronics). Bright field and fluorescence images were acquired with an Olympus DT70 CCD color camera.
3
Immunocytochemistry of Connexin-43 in Glioblastoma
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Differentiated glioblastoma cells were seeded in 24-well plates (Sarstedt, Nümbrecht, Germany) with a density of 1500 cells/well, on glass coverslips (Langenbrinck GmbH, Emmendingen, Germany). Cells were fixed with 4% paraformaldehyde at room temperature (RT) for 20 min. The cells were then permeabilized with 20% Methanol for 15 min. To block unspecific binding of antibody, cells were then incubated with 20% Bovine serum albumin (BSA) at RT for 60 min. Primary antibody mix (200 μL; rabbit polyclonal anti-connexin-43 (Sigma, St. Louis, MO, USA) and rabbit monoclonal ß-tubulin (Cell signaling, Cambridge, UK), dilution 1:1000) was added to each coverslip containing wells and incubated at 37 °C for 60 min. Incubation with secondary antibodies (goat anti-rabbit Alexa Fluor 488 and Alexa Fluor 647 (Sigma, St. Louis, MO, USA), dilution 1:1000) was performed at RT for 60 min. DAPI Fluoromount-G® mounting medium (SouthernBiotech, Birmingham, AL, USA) was used for nuclei staining. Images were taken with the use of AX 70 microscope and processed with Zeiss Zen software (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). Fluorescence intensity was calculated as follows: integrated density—(Area of selected cells × mean fluorescence of background).
4
Immunocytochemistry of Cell-Cell Junctions
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Immunostaining was performed as previously described [4 (link)]. Briefly, cells were seeded on glass coverslips coated with laminin. Then, cells were fixed with 4% paraformaldehyde, permeabilized with 20% methanol followed by incubation with a primary antibody mix of Cx43 and ß-tubulin and the corresponding secondary antibodies. In addition, the cell nuclei were stained with DAPI Fluoromount mounting medium. The immunofluorescence images were acquired with an AX70 microscope from Zeiss and evaluated with the Zeiss Zen Blue software.
5
Immunofluorescence staining of PCa cells
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For staining with primary and secondary antibodies, alternatively, cell suspensions were fixed on slides in methanol in −20 °C for 10 min. PCa cells were grown on glass coverslips in phenol red‐free RPMI‐1640 medium containing 10% FBS for 24 h and fixed with 4% paraformaldehyde in PBS. The slides were stained with primary antibodies. Primary antibodies including anti HLA‐ABC conjugated with FITC and anti‐FCGR3A (CD16a), was purchased from Biosite (Bioss MA, USA), and PIP5K1α (Protein Technologies, UK) was used. Secondary antibodies include anti‐rabbit conjugated to Alexa Fluor 488 (Invitrogen, Stockholm, Sweden), anti‐mouse conjugated to Alexa Fluor 546 (Invitrogen, Stockholm, Sweden), and anti‐rabbit conjugated to Rhodamine (Chemicon International Inc, Temecula, CA). Cells were counterstained with DAPI (4',6‐diamidino‐2‐phenylindole, dihydrochloride) (SERVA Electrophoresis GmbH, Heidelberg, Germany). The cells were examined under an Olympus AX70 microscope using NIS Elements f 2.20 software or a Zeiss Apoptome microscope (Zeiss, Germany) and the zen 2.3 lite software (Zeiss, Germany).
6
Immunofluorescence Staining of Cellular Proteins
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Cell suspensions were fixed on slides in methanol or in 4% paraformaldehyde in PBS. The slides were washed in PBS twice and permeabilized in 0.5% Triton X-100 (Sigma-Aldrich, Stockholm, Sweden) for 10 min at room temperature (RT). The slides were stained with primary antibodies. Primary antibodies including anti-PIP5K1α (Protein Technologies, Manchester, UK) and β-tubulin (Abcam, Cambridge, UK) were used. Secondary antibodies including anti-rabbit conjugated to Alexa Fluor 488 (Invitrogen, Stockholm, Sweden), anti-mouse conjugated to Alexa Fluor 546 (Invitrogen, Stockholm, Sweden) and anti-rabbit conjugated to Rhodamine (Chemicon International Inc, Temecula, CA, USA) were used. Cells were counterstained with DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride). The cells were examined under an Olympus AX70 microscope using the NIS Elements F 2.20 software or a Zeiss Apotome microscope and the Zen 2.3 Lite software (Zeiss, Oberkochen, Germany).
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