The GECs on insert filters were fixed in 4% paraformaldehyde for 20 min and blocked by 5% BSA for 2 h at room temperature. After washed with PBS for 3 times, GECs were incubated by primary antibodies of ZO-1, occludin, and claudin-5 (1:50; Life Technologies) at 4°C overnight. The nuclei were stained by DAPI (0.5 mg/ml, Beyotime Institute of Biotechnology) for 8 min. The fluorescence was visualized by DP71 immunofluorescence microscope (Olympus, Tokyo, Japan), and merged by Chemi Imager 5500 V2.03 software.
Dp71 immunofluorescence microscope
Manufactured by Olympus
Sourced in Japan
The DP71 immunofluorescence microscope is a high-performance microscope designed for imaging and analysis of fluorescently labeled samples. It features a high-resolution digital camera and advanced imaging software to capture detailed images of cellular structures and biomolecular interactions.
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4 protocols using dp71 immunofluorescence microscope
1
Immunostaining of Tight Junction Proteins
2
Evaluating Angiogenic Potential of Cancer Cells
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The 96-well plates were coated with 100 μL Matrigel (BD Biosciences, Bedford, MA, United States) per well and maintained at 37°C for 30 min. Thereafter, HUVEC cells were seeded in the plates at a density of 1 × 104 cells/well. After the cells were fully adhered, the medium was replaced with conditioned medium from Caki-1 and 786-O cells. After 24 h of incubation, the cells were monitored and imaged using an Olympus DP71 immunofluorescence microscope (Olympus, Tokyo, Japan), and the total length and branch number of the tubules in the focused area were measured and analyzed using the Chemi Imager 5500 V2.03 software (Alpha Innotech, San Leandro, CA, United States) (14 (link), 23 (link)).
3
In Vitro Tubule Formation Assay
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To determine the angiogenic capacity of endothelial cells, a tubule-formation assay was performed in vitro. Pre-coated Matrigel (5 mg/mL, BD, USA) was solidified via incubation on a 48-well plate at 37 °C for 30 min. The fused HUVEC cells were suspended in the culture medium at a density of 1.5×105 cells/mL and incubated at 37 °C for 24 h. Photographs were then taken with an Olympus DP71 immunofluorescence microscope and the number of microtubules was determined.
4
Vasculogenic Potential of Gastric Cancer
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A 96-well plate was coated with Matrigel (BD Biosciences; 100 μL/well), followed by a 30-min standing at 37ºC. Afterwards, human umbilical vein endothelial cells (HUVECs) were seeded into the plates at a density of 1 × 104 cells/well. After the cells fully adherent to the wells, the medium was replaced with a conditioned medium of AGS and MKN-45 cells. After 24-h incubation, cells were monitored and imaged using an Olympus DP71 immunofluorescence microscope (Olympus, Tokyo, Japan), and the number of microvessels in the focus area was measured and analyzed using Chemi Imager 5500 V2.03 software (Alpha Innotech, San Leandro, CA, USA).
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