An immunofluorescence microscopy-based internalisation assay was performed on HEK293-rsst2 cells, as previously described [22 (link)]. Briefly, the cells were treated with different antagonist chelator-peptide conjugates and/or [Tyr3]octreotide (TOC) (the sst2 agonist) at 37 °C for 30 min. After fixation and permeabilisation, cells were stained with the sst2-specific primary antibody R2-88 (provided by Dr. Agnes Schonbrunn, McGovern Medical School, University of Texas Health Science Center, Houston, Texas, USA) as described previously [25 (link), 26 (link)]. The cells were imaged using a Leica DM RB immunofluorescence microscope, and the images were acquired using an Olympus DP10 camera.
Dp10 camera
Manufactured by Olympus
Sourced in Japan
The DP10 camera is a digital microscope camera designed for laboratory use. It captures high-quality images and video to document observations and analyses. The DP10 provides a reliable way to record data from microscopic samples.
Automatically generated - may contain errors
Lab products found in correlation
Yo pro 1, by Thermo Fisher Scientific (1 mentions)
Dm2500 microscope, by Olympus (1 mentions)
Envision dual link system hrp, by Agilent Technologies (1 mentions)
Ax70 microscope, by Olympus (1 mentions)
Anti aqp1, by Abcam (1 mentions)
Bx50 fluorescence microscope, by Olympus (1 mentions)
Dioc18 3, by Thermo Fisher Scientific (1 mentions)
Bx40 microscope, by Olympus (1 mentions)
7 protocols using dp10 camera
1
Internalization Assay for Somatostatin Receptor 2
2
Labeling Zebrafish Hair Cells with YO-PRO-1
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Neuromast hair cells in zebrafish larvae were labeled with fluorescent dye YO-PRO-1 (Thermo Fisher Scientific, USA), which can stain cellular nuclei by binding to DNA. Zebrafish larvae (four replicates, three larvae each) were incubated in 0.3×Danieau’s solution containing 3 μM YO-PRO-1 for 15 min, rinsed three times with 0.3×Danieau’s solution, and then anesthetized with 0.001 % buffered MS-222. The labeled hair cells in otic neuromasts were observed under a Leica DM2500 microscope with a green fluorescence filter set and an Olympus DP10 camera.
3
Immunohistochemical Analysis of AQP1 Expression
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All samples examined were obtained from formalin-fixed, paraffin-embedded pieces. Slices of 5 μm were cut with a microtome and mounted on microscope slides. Immunohistochemical samples were obtained from paraffin sections that were immersed in xylene and rehydrated through a series of decreasing dilutions of ethanol. Blocking of endogenous peroxidase, epitope retrieval and immunostaining procedure were done as previously reported (López-Campos et al., 2011 (link)). Primary antibody, rabbit polyclonal anti-AQP1, (1:500 dilution, Abcam, Cambridge, UK) was used overnight, for the developing of brown precipitates we then used the two steps system EnVision + Dual Link System-HRP (DakoCytomation, Dako Denmark). Qualitative analysis was made by two independent observers. We assigned zero for no staining on the sample, low positive (<25% of the sample stained), medium positive (50% of the sample stained) and high positive (>75% of the sample stained) values (Tables 1 , 2 ). Immunoreactivity was analyzed in terms of surface and type of cells showing AQP1 on their surface. Samples were photographed using an AX70-Olympus microscope equipped with an Olympus DP10 camera (Denmark).
4
Microbial Viability Visualization
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In order to visualise the viable and dead microbial cells, the LIVE/DEAD R ⃝ BacLight Bacterial Viability Kit was used, which was supplied by Invitrogen (ThermoFisher Scientific, UK). This kit contains two fluorescent reagents: (i) SYTO 9 (green-fluorescent nucleic acid stain) that marks all microbial cells with either intact or damaged membranes; (ii) propidium iodide (red-fluorescent nucleic acid stain), which penetrates exclusively the microorganisms with injured membranes to reduce SYTO 9 stain fluorescence when both are present.
The dye components were mixed at a ratio of 1:1 and 0.4 µL of the blend was incorporated into 250 µL of each microbial suspension. Subsequently, the preparation was mixed and incubated at room temperature for 10 min to avoid coming into contact with light. Then 5 µL of the stained cells were laid on poly-L-lysine-covered slides (Sigma-Aldrich, Madrid, Spain). A coverslip was placed over the suspension and sealed. Finally, slides were visualised under an Olympus BX 50 fluorescence microscope with a U-MWB double filter and an Olympus DP-10 camera.
The dye components were mixed at a ratio of 1:1 and 0.4 µL of the blend was incorporated into 250 µL of each microbial suspension. Subsequently, the preparation was mixed and incubated at room temperature for 10 min to avoid coming into contact with light. Then 5 µL of the stained cells were laid on poly-L-lysine-covered slides (Sigma-Aldrich, Madrid, Spain). A coverslip was placed over the suspension and sealed. Finally, slides were visualised under an Olympus BX 50 fluorescence microscope with a U-MWB double filter and an Olympus DP-10 camera.
5
Fluorescent Membrane Dye Imaging
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Cells were stained with fluorescent lipophilic membrane dyes (tracers) such as the red-orange fluorescent DilC18(3) (1,1'-Dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate; maximum fluorescence excitation 549 nm and emission 565 nm; Thermo Fisher Scientific, USA) or the green fluorescent DiOC18(3) (3,3′-Dioctadecyl oxacarbocyanine perchlorate; maximum fluorescence excitation 484 nm and emission 590 nm; Thermo Fisher Scientific, USA) dissolved in DMSO according to seller's instructions. At intervals of 3 days, cultured specimens were observed under an inverted fluorescence microscope (IM 35, Zeiss) equipped with proper excitation and emission filters according to the intravital stain used, and digitally photographed with a DP10 Camera (Olympus, Japan).
6
Megakaryocyte Colony Forming Assay
7
Histopathological Analysis of Animal Paws
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After the sacrifice, animal paws were collected, fixed in 10% buffered formalin and decalcified with 10% formic acid to undergo histopathological analysis. In order to evaluate the surroundings of the site of application, fixed decalcified paws were processed for embedding in paraffin wax by using routine protocol. Sections (5 μm thick) were stained with haematoxylin and eosin (H&E). The slides were examined using light microscopy using an Olympus BX 40 microscope coupled with an Olympus DP 10 camera (Olympus, Shinjuku, Tokyo, Japan). Digital photographs were taken at an amplification of 100×, except for the control that was acquired at an amplification of 40×. The histological samples were evaluated for synovial inflammation and bone erosion. Synovial inflammation was scored as follows: 0- no inflammation; 1- slight synovitis with some cell infiltration; 2- moderate synovitis with moderate cell infiltration; 3- extensive synovitis with a moderate number of infiltrating cells; 4- extensive and severe synovitis, with the presence of numerous inflammatory cells. Bone erosion was scored as follows: 0- no erosion; 1- small areas of resorption; 2- numerous areas of resorption; 3- extensive osteolysis; 4- extensive and severe osteolysis.
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