Mouse WATs were isolated and fixed in 4% PFA followed by embedding in paraffin. Paraffin-embedded tissues were sectioned and stained with hematoxylin–eosin (HE). The immunostaining was performed as previously described [39 (link)]. Sections were incubated at 4°C overnight with primary antibodies. Histology or immunostained samples were viewed under microscope (IX71; OLYMPUS, Tokyo, Japan) with a UPlan-FLN 4× objective/0.13 PhL, a UPlan-FLN 10× objective/0.30 PhL, or a LUCPlan-FLN 20× objective/0.45 PhL. Images were captured with a digital camera (IX-SPT; OLYMPUS, Tokyo, Japan) and Digital Acquire software (DPController; OLYMPUS, Tokyo, Japan). WATs were viewed under microscope (SZX16; OLYMPUS, Tokyo, Japan) with an SDF-PLAPO 1× PF. Images were captured with a digital camera (U-LH100HGAPO; OLYMPUS, Tokyo, Japan) and Digital Acquire software (DPController; OLYMPUS, Tokyo, Japan).
Dp controller
Manufactured by Olympus
Sourced in Japan, United States
The DP Controller is a digital processing unit designed to interface with Olympus digital cameras and microscopes. It provides control and data transfer functionalities to support advanced imaging applications. The DP Controller enables seamless integration between Olympus imaging hardware and software solutions.
Automatically generated - may contain errors
Lab products found in correlation
Dp manager, by Olympus (12 mentions)
Bx51 microscope, by Olympus (3 mentions)
Hoechst 33342, by Merck Group (3 mentions)
Ix71 fluorescent microscope, by Olympus (3 mentions)
Leukocyte acid phosphatase kit, by Merck Group (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Ix81 motorized inverted microscope, by Olympus (2 mentions)
Mz6 microscope, by Olympus (2 mentions)
Sc 462 r20, by Santa Cruz Biotechnology (2 mentions)
Fluoromount plus dapi, by Vector Laboratories (2 mentions)
Dp manager software, by Olympus (2 mentions)
Ix spt, by Olympus (2 mentions)
U lh100hgapo, by Olympus (2 mentions)
Szx16, by Olympus (2 mentions)
Intravital microscope, by Olympus (2 mentions)
Model bx51wi, by Olympus (2 mentions)
Model dp71, by Olympus (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Hrp labeled secondary antibody, by ZSGB-BIO (1 mentions)
Horseradish peroxidase, by ZSGB-BIO (1 mentions)
69 protocols using dp controller
1
Histological and Immunostaining Analysis of Mouse WATs
2
Immunohistochemistry and Immunofluorescence Assays
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Immunohistochemistry on 5-μm paraffin sections using antibodies against Ki-67 (Novocastra Laboratories), cleaved caspase-3 (Cell Signaling), CD31 (Abcam), α1(IV) (Abgent) or DDR1 (Santa Cruz) was performed as described [40 (link)]. For α5(IV) immuno-staining, 8-μm frozen tissue sections were fixed in cold acetone for 10 min. Samples were incubated with α5(IV) antibody (rat monoclonal antibody clone b14) (1:50–1:100) for 16 hours at 4°C, followed by incubation with Alexa Fluor 555/488 conjugated anti-rat IgG antibody. Immunohistochemistry or immunofluorescence sections were viewed under microscope (IX71; OLYMPUS, Inc.) with a UPlan-FLN 4×objective/0.13 PhL, a UPlan-FLN 10×objective/0.30 PhL, or a LUCPlan-FLN 20×objective/0.45 PhL. Images were captured with a digital camera (IX-SPT; OLYMPUS, Inc.) and Digital Acquire software (DPController; OLYMPUS, Inc.). Perfused blood vessels in Matrigel plugs were viewed by UV-illumination under microscope (SZX16; OLYMPUS, Inc.) with a SDF-PLAPO 1×PF. Images were captured with a digital camera (U-LH100HGAPO; OLYMPUS, Inc.) and Digital Acquire software (DPController; OLYMPUS, Inc.).
3
Immunohistochemical Staining of Colorectal Tumors
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The tumors were harvested, formalin-fixed, paraffin-embedded, and made into slides. Then, the slides were deparaffinized in xylene and then were rehydrated through an ethanol gradient. The sections were heated at 100°C in 0.01 mol/L sodium citrate buffer (pH 6.0) for 10 min for antigen retrieval. The slides were inactivated with 3% H2O2 for 15 min at room temperature in the dark place, blocked with 10% goat serum for 10 min at 37°C, and incubated with 100 µl primary antibodies diluted in PBS to each section overnight at 4°C, 100% humidity. Next day, the sections were stained with horseradish peroxidase (HRP)–labeled secondary antibody (ZSGB-BIO, Beijing, China) at 37°C for 40 min. Finally, the sections were incubated with 3,3′-diaminobenzidine (DAB, ZSGB-BIO, Beijing, China, and ZLI-9019) to perform color development. After counterstaining with hematoxylin, the sections were observed and recorded using a microscope with an Olympus DP controller (Olympus). A staining score was calculated by multiplying two scores of staining intensity (0: negative staining; 1: weak staining; 2: moderate staining; 3: strong staining) and intensity (0: 0–25%; 1: 25–50%; 2: 50–75%; 3: >75%) of positive stained colorectal cancer cells.
4
Quantitative Analysis of Nerve Fibers
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Image analysis software Olympus DP Controller and DP Manager (Olympus Corp., Toyko, Japan) was used with 400-power random observation for 50 visual fields. All the immunoreactive fibers were numbered and the density of the nerve fibers was calculated (fibers/cm2). The data collection was completed by two pathological physicians in a double-blind manner.
5
Osteoclast Differentiation and Quantification
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MNCs were prepared with 4% paraformaldehyde for 5 min. on day 4 of differentiation. Fixed cells were made permeable with 0.1% Triton X-100 for 5 min. and stained for TRAP using the Leukocyte Acid Phosphatase Kit (Sigma-Aldrich, St. Louis, MO, USA). Images of TRAP-positive cells under the microscope were captured with DP Controller (Olympus Optical, Japan). Also, to investigate CZ's effect on osteoclast formation in CIA mice, section slides were stained using a TRAP staining kit. TRAP-positive cells in ten areas of each ankle were counted.
6
ROS Detection in Cell Cultures
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The generation of intracellular ROS was examined using the ROS detection reagent according to manufacturer instructions. Briefly, cells were grown in a 96-well black plate (Eppendorf Ltd., Germany) and subjected to different treatments with/without TPP-Niacin and H2O2. Then, the cells were incubated with 5 μM H2DCF-DA or 10 μM DHE at 37 °C for 20 min. The fluorescence intensities were measured using the fluorescence plate reader (Bio-Tek) at Ex./Em. = 495/527 nm for H2DCF-DA and Ex./Em. = 535/610 nm for DHE. Then, the H2DCF-DA-stained cell images were obtained with a fluorescent microscope IX51 with DP controller (Olympus Optical, Japan). All samples were prepared in triplicate, and each experiment was repeated three times.
7
Quantifying Blood and Lymphatic Vessels in Uterine Tissues
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Each slide was examined at ×200 magnification and images captured with the use of an Olympus BX51 microscope and a DP70 digital camera with DP controller and DP manager (Olympus Optical Co., LTD, Tokyo, Japan; Figure 1(a)–(h) ). Assessments of blood and lymphatic densities were performed on 15 non-overlapping images where possible.
Blood and lymphatic vessels were counted manually for each image taken by two independent observers (N.P.N., F.M.) without knowledge of the phase of the menstrual cycle or other clinical parameters. Concordance between the two observers was calculated for the average of all fields of view.
The interclass correlation coefficients (ICCs) for inter-rater reliability were good. For polyp blood vessel density, the ICC was 0.994 (95% confidence interval (CI) 0.985–0.998; n = 20, p < 0.001), for adjacent endometrium 0.989 (95% CI 0.972–0.996; n = 8, p < 0.001), for distant endometrium 0.993 (95% CI 0.978–0.992; n = 14, p < 0.001) and for control endometrium 0.990 (95% CI 0.991–0.996, n = 32, p < 0.001).
The ICC for polyp lymphatic density was 0.991 (95% CI 0.993–0.998; n = 17, p < 0.001), for adjacent endometrium 0.986 (95% CI 0.981–0.993; n = 7, p < 0.001), for distant endometrium 0.996 (95% CI 0.987–0.995; n = 13, p < 0.001) and for control endometrium 0.993 (95% CI 0.991–0.997; n = 31, p < 0.001).
Blood and lymphatic vessels were counted manually for each image taken by two independent observers (N.P.N., F.M.) without knowledge of the phase of the menstrual cycle or other clinical parameters. Concordance between the two observers was calculated for the average of all fields of view.
The interclass correlation coefficients (ICCs) for inter-rater reliability were good. For polyp blood vessel density, the ICC was 0.994 (95% confidence interval (CI) 0.985–0.998; n = 20, p < 0.001), for adjacent endometrium 0.989 (95% CI 0.972–0.996; n = 8, p < 0.001), for distant endometrium 0.993 (95% CI 0.978–0.992; n = 14, p < 0.001) and for control endometrium 0.990 (95% CI 0.991–0.996, n = 32, p < 0.001).
The ICC for polyp lymphatic density was 0.991 (95% CI 0.993–0.998; n = 17, p < 0.001), for adjacent endometrium 0.986 (95% CI 0.981–0.993; n = 7, p < 0.001), for distant endometrium 0.996 (95% CI 0.987–0.995; n = 13, p < 0.001) and for control endometrium 0.993 (95% CI 0.991–0.997; n = 31, p < 0.001).
8
Coumarin-6 Loaded SLN Uptake
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SLNs containing coumarin-6 were synthesized using the same method as mentioned above. Synthesis was carried out in the dark. To remove free coumarin-6 from the SLN suspension, SLN-coumarin-6 were centrifuged at 20,000× g, 30 min (Hettich universal 320R) and SLN-coumarin-6 pellet was resuspended in ultra-pure water. Cells were seeded at a density of 30,000 cells/mL in a Petri dish and allowed to adhere overnight at 37 °C, 5% CO2. They were treated with SLN-coumarin-6 and visualized by using fluorescent microscopy (Olympus BX51; software DPController, Olympus Optical) at time points 1 h, 3 h, 6 h, 24 h and 48 h.
9
Adipose-Derived Stem Cell Differentiation
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ADSCs were seeded into a 24-well plate (5×103 cells/well) and cultured in basic medium (DMEM plus 10% FBS) for 1 day. Then, the cells were incubated with DFC-CM for another 7 days. They were then fixed in 4% paraformaldehyde for 15 min, after which the cells were incubated with β-catenin (Abcam,32572), CAP (cemetum attachment protein) or BSP (bone sialoprotein) at dilutions ranging from 1∶100 to 1∶200 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 2 h and subsequently incubated with FITC(Fluorescein Isothiocyanate)- or Rhodamine-conjugated anti-rabbit or anti- goat secondary antibodies. Then, sections were counterstained with Hoechst 33342 (5 µg/mL; Sigma) to identify all nuclei. The image collection and superimposition were processed by DP controller (Olympus Optical, Tokyo, Japan) and DP manager (Olympus Optical, Tokyo, Japan). Isotype-matched control antibodies were used under the same conditions.
10
Quantifying Mature Osteoclasts via TRAP Staining
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For measuring TRAP activity, cells were fixed in 10% formalin for 10 min., rinsed in PBS for 3 changes and stained using a Leukocyte Acid Phosphatase kit (Sigma‐Aldrich). Images of TRAP‐positive cells were captured under a microscope with a DP Controller (Olympus Optical, Tokyo, Japan). The number of mature osteoclasts was quantified by counting the number of multinucleated TRAP+ cells (>3 nuclei) in a representative area, in each of the three replicate samples.
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