Paraffin sections (5 μm) were deparaffinized and hydrated to deionized water. Next, slides were immersed in periodic acid solution (Sigma-Aldrich, St. Louis, MI, USA) for 5 min at room temperature. Then, the slides were rinsed several times in distilled water and immersed in Schiff’s Reagent (Sigma-Aldrich, St. Louis, MI, USA) for 15 min. At the end of the incubation, slides were washed in running tap water for 5 min, dehydrated, mounted with ROTI®Histokitt (Carl Roth GmbH, Karlsruhe, Germany), and imaged on the Olympus IX2-UCB microscope.
Ix2 ucb microscope
Manufactured by Olympus
Sourced in Germany, United States
The IX2-UCB is a compact and versatile upright microscope designed for a wide range of applications. It features a robust and stable frame, allowing for precise and reliable observations. The microscope is equipped with an infinity-corrected optical system, ensuring high-quality imaging and efficient light transmission. The IX2-UCB is a reliable and versatile tool for various research and educational purposes.
Automatically generated - may contain errors
Lab products found in correlation
Roti histokitt, by Carl Roth (2 mentions)
Periodic acid solution, by Merck Group (1 mentions)
Schiff s reagent, by Merck Group (1 mentions)
Biocoat matrigel invasion chamber, by Corning (1 mentions)
Motorized stage, by MBF Biosciences (1 mentions)
Dylight 488, by Vector Laboratories (1 mentions)
Alexa 594, by Abcam (1 mentions)
Lsm710nlo confocal laser scanning microscope, by Zeiss (1 mentions)
Em ccd camera, by Hamamatsu Photonics (1 mentions)
Alexa 488, by Abcam (1 mentions)
Dylight 594, by Vector Laboratories (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Alizarin red, by Olympus (1 mentions)
Alizarin red, by Merck Group (1 mentions)
Eosin solution, by Merck Group (1 mentions)
Fv3000, by Olympus (1 mentions)
Pannoramic desk, by 3DHISTECH (1 mentions)
9 protocols using ix2 ucb microscope
1
Periodic Acid-Schiff Staining Protocol
2
Invasion Assay of Human OS 143B Cells
3
Quantifying Cell Invasion via Matrigel Chambers
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Cells were seeded in 6-well plates and transfected with 10 nM of htRNALeu/miR-34a-5p, htRNALeu/miR-124-3p or htRNALeu. After 48 h, survived cells were harvested and seeded onto the upper inserts of the 24-well Corning BioCoat Matrigel Invasion Chambers (Corning, Bedford, MA) at 6 × 104 cells/well with 500 μL serum-free RPMI 1640 medium where the lower chamber was supplied with 750 μL of serum-containing culture medium (10% FBS). 20 h later, the invaded cells were fixed with 500 μL of 10% formaldehyde, stained with 500 μL 0.1% crystal violet, and imaged with an Olympus IX2-UCB microscope. The number of invaded cells was acquired by counting five fields per insert (100 × magnification), and then compared between different treatments as we described recently 31 (link).
4
Multiparametric Immunofluorescence Assay
5
Evaluating Invasive Potential of Cancer Cells
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Invasive potential of 143B-GFP-Luc cells, treated with vehicle, or 12 nM miR-34a, or 40 nM doxorubicin plus 4 μM sorafenib, or the combination for 72 h, was assessed as described previously [16 (link), 17 (link), 45 (link)] by using Corning BioCoat Matrigel Invasion Chamber with 8.0 mm PET membrane coated with matrigel (Corning, NY, USA). Briefly, 5 × 104 cells in 500 μl serum-free RPMI 1640 media were placed into the upper insert, and 750 μl medium supplemented with 10% FBS was added into the well as a chemo-attractant. 30 h later, cells in the upper inserts were removed and the lower were fixed with 10% formalin, stained with 0.1% crystal violet, and then photographed with five fields per insert under an Olympus IX2-UCB microscope (40 × magnifications). Each treatment group was conducted in triplicate and assessed independently. The quantification of invaded cells was calculated using Image J software and invasion capacity was determined by adjusting the vehicle control group to 100%.
6
Quantifying Cell Invasion Potential
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Cells were treated with 4 nM tRNA/mir-34a or tRNA/MSA, or vehicle. After 72 h, 3 × 104 cells were subjected to invasion assay using the Corning BioCoat Matrigel Invasion Chamber with 8.0 μm PET membrane (Corning, NY) for 24 h. The upper inserts with matrigel coating were then fixed with 10% formalin, stained with 0.1% crystal violet, and photographed under an Olympus IX2-UCB microscope (200× magnification). Cells were treated in triplicate and assayed separately. Five fields per insert were photographed. The number of cells was counted using Adobe Photoshop’s count tool, and compared between different treatments as we described recently50 (link).
7
Alizarin Red Staining and Calcium Quantification
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Cells were fixed with 10% formalin solution for 20 min at room temperature (RT) and rinsed with PBS. 40 mM solution of Alizarin Red (catalogue N 5533-25G, Sigma-Aldrich) was added to the cells for 30 min at RT with agitation. Next, unbound Alizarin Red dye was removed, and the cells were washed several times with Milli-q water. Alizarin Red staining was detected by the Olympus IX2-UCB microscope in the phase-contrast setting.
For calcium quantification, 10% acetic acid (v/v) was added to the cells stained with Alizarin Red and incubated for 30 min at RT with agitation. Cells were scraped off, transferred to Eppendorf tubes, vortexed for 30 s, and incubated for 10 min at 85 °C. Next, samples were centrifuged for 15 min at 16 g and 200 μl of the supernatant was transferred to another Eppendorf tube. 22.5 μl of 10% NH4OH (v/v) was added to the samples and mixed. The absorbance was measured at 405 nm. Values were normalized to a calibration curve.
For calcium quantification, 10% acetic acid (v/v) was added to the cells stained with Alizarin Red and incubated for 30 min at RT with agitation. Cells were scraped off, transferred to Eppendorf tubes, vortexed for 30 s, and incubated for 10 min at 85 °C. Next, samples were centrifuged for 15 min at 16 g and 200 μl of the supernatant was transferred to another Eppendorf tube. 22.5 μl of 10% NH4OH (v/v) was added to the samples and mixed. The absorbance was measured at 405 nm. Values were normalized to a calibration curve.
8
Paraffin Sectioning and H&E Staining
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Paraffin sections (5 μm) were deparaffinized, hydrated, and stained in Gill’s haematoxylin solution 2 (Sigma-Aldrich) for 3 min. After that, the slides were washed in running tap water for 5 min, rinsed in distilled water, and dipped in 95% ethanol. Next, the eosin co-staining was performed by immersing slides in the eosin solution (Sigma-Aldrich) for 1 min. Stained slides were washed in 95% ethanol, dehydrated, and mounted with ROTI®Histokitt (Carl Roth). Images were acquired on the Olympus IX2-UCB microscope.
9
Microscopy Imaging Protocol Workflow
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Unless specifically mentioned, bright-field images were taken with an Olympus IX2-UCB microscope, and fluorescence images were taken with an Olympus FV3000 confocal laser-scanning microscope. Tissue sections are imaged by a slide scanner (Pannoramic DESK, 3DHISTECH). Images were analyzed using Image J (NIH).
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