Immunofluorescence staining for pimonidazole was performed as previously described [39 (link)] on 5µm thick xenograft cryosections. Tumor sections were double stained for pimonidazole and pSMAD2 or pSMAD3 antibody (1:50). For quantitation of pSMAD staining intensity, a minimum of 6 representative areas from at least 3 different tumors for each condition were captured using an Axioskop 2 phase-contrast/epifluorescence microscope (Carl Zeiss Inc., Oberkochen, Germany). Fluorescence intensities were analyzed using the ImagePro Plus software version 6 (Media Cybernetics, Rockville, MD, USA). The sum of pSMAD labeling intensity (density) relative to the total area of pimonidazole positive or negative areas was calculated.
Axioskop 2 phase contrast epifluorescence microscope
Manufactured by Zeiss
Sourced in United States
The Axioskop 2 is a phase-contrast/epifluorescence microscope manufactured by Zeiss. It is designed for high-resolution imaging of cell cultures and tissue samples. The microscope features a motorized stage, plan-apochromatic objectives, and an LED illumination system. It supports both phase-contrast and epifluorescence imaging techniques.
Automatically generated - may contain errors
Lab products found in correlation
Image pro plus software version 6, by Media Cybernetics (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Oregon green 488 conjugated gelatin, by Thermo Fisher Scientific (1 mentions)
Diaminobenzidene, by Vector Laboratories (1 mentions)
4 protocols using axioskop 2 phase contrast epifluorescence microscope
1
Quantifying Hypoxia-Induced pSMAD Signaling
2
Hypoxia and Vasculature Labeling in CAM Tumors
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CAMs bearing tumors were intravenously injected with pimonidazole hydrochloride (1.5 mg/CAM) to detect hypoxia and Lens culinaris agglutinin (LCA) (0.05 mg/CAM) to label the CAM blood vessel network [40 (link),41 (link),42 (link),43 (link),44 (link)], 30 min before harvesting tumors. Tumors removed from the CAM were placed directly in the cryopreservative embedding media OCT compound (Electron Microscopy Sciences, Hatfield, PA, USA, 62550-01), immediately frozen in dry ice-cooled isopentane and kept at −80 °C until sectioning for immunostaining. Sections were fixed with PFA 2% for 10 min at 4 °C. Blocking and staining were performed in BSA 2% with 10% goat serum. Pimonidazole was detected with mouse antibody (Hypoxyprobe, 1:200) and goat anti-mouse Alexa Fluor 488 (Invitrogen). Nuclei were stained with 1 µg/mL DAPI (4,6-diamidino-2-phenylindole, Invitrogen, Molecular Probes, Eugene, OR, USA). Stained sections were visualized using an Axioskop 2 phase-contrast/epifluorescence microscope (Carl Zeiss Inc., Thornwood, NY, USA).
3
Quantifying ECM-Degrading Invadopodia
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Coverslips were prepared as previously described [50 (link)], using Oregon-Green488-conjugated gelatin (Invitrogen, Burlington, ON, Canada). Forty thousand cells were seeded on each coverslip and allowed to adhere. Following various incubation times as described within the figure legends, cells were fixed with 2% paraformaldehyde for 10 min at room temperature. Nuclei were stained with DAPI and F-actin was stained using Texas-Red-conjugated phalloidin. Cells were visualized and imaged by fluorescence microscopy using an Axioskop 2 phase-contrast/epifluorescence microscope (Carl Zeiss, Inc., Thornwood, NY, USA). Cells forming ECM-degrading invadopodia were identified based on cells with at least one F-actin-enriched area of matrix degradation (characterized by loss of green fluorescence). Three fields of 100 cells (magnification 40×) were counted per coverslip to quantify the percentage of cells forming ECM-degrading invadopodia.
4
Immunohistochemical Analysis of Synovial Tissue
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Tissue sections from human joints or the left hind knee joints of rats were processed immediately after excision, after a standardized paraffin embedding protocol. Tissue sections were rehydrated and treated with 1% trypsin (rat tissues) or 0.01 mol/L citrate, pH 6.0 (human tissues), and immunohistochemical staining was performed according to the standard avidin-biotin immunoperoxidase complex technique using primary antibodies (1:50 dilution in 2% bovine serum albumin) or isotype-matched negative controls and diaminobenzidene (Vector Laboratories Inc., Burlingame, CA) as substrate. For quantitative immunohistochemistry, photomicrographs of three representative areas of the synovial membrane for each joint section were captured using an Axioskop 2 phase-contrast/epifluorescence microscope (Carl Zeiss Inc., Thornwood, NY) equipped with a 10Â objective and a Retiga SRV cooled color digital camera (Qimaging, Surrey, BC, Canada). Images were analyzed as previously described. 10
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