PP-containing small intestines were fixed in 10% formalin, routinely processed, and embedded in paraffin. Sections were stained with hematoxylin and eosin (H&E). For immunofluorescence staining, fixed, frozen small intestinal sections were rehydrated in PBS for 30 minutes and permeabilized in 0.2% Triton X-100/PBS for 10 minutes. The slides were blocked with 1% BSA/PBS for 1 hour at room temperature and incubated with Alexa Fluor 488 wheat germ agglutinin (WGA, Molecular Probes) and Alexa Fluor 568 Phalloidin (Molecular Probes) for 30 minutes. Sections were washed 3 times with PBS after each antibody incubation. After a 10-minute stain with DAPI, slides were mounted using Prolong Gold antifade (Molecular Probes). Sections were visualized using the BX51 microscope, DP72 camera, and DP2-BSW imaging software (Olympus America Inc.). WGA-positive luminal signals were quantified using Imaris software (Bitplane).
Dp2 bsw imaging software
Manufactured by Olympus
Sourced in Japan
DP2-BSW is an imaging software developed by Olympus. It provides basic functionality for acquiring, displaying, and managing digital microscope images.
Automatically generated - may contain errors
Lab products found in correlation
Bx51 microscope, by Olympus (4 mentions)
Dp72 camera, by Olympus (3 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 568 phalloidin, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 wheat germ agglutinin wga, by Thermo Fisher Scientific (2 mentions)
Epidermal growth factor (egf), by Merck Group (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Noble agar, by Merck Group (1 mentions)
Dp 72, by Olympus (1 mentions)
6 protocols using dp2 bsw imaging software
1
Histological and Immunofluorescence Analysis of Small Intestine
2
Histological and Immunofluorescence Analysis of Small Intestine
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PP-containing small intestines were fixed in 10% formalin, routinely processed, and embedded in paraffin. Sections were stained with hematoxylin and eosin (H&E). For immunofluorescence staining, fixed, frozen small intestinal sections were rehydrated in PBS for 30 minutes and permeabilized in 0.2% Triton X-100/PBS for 10 minutes. The slides were blocked with 1% BSA/PBS for 1 hour at room temperature and incubated with Alexa Fluor 488 wheat germ agglutinin (WGA, Molecular Probes) and Alexa Fluor 568 Phalloidin (Molecular Probes) for 30 minutes. Sections were washed 3 times with PBS after each antibody incubation. After a 10-minute stain with DAPI, slides were mounted using Prolong Gold antifade (Molecular Probes). Sections were visualized using the BX51 microscope, DP72 camera, and DP2-BSW imaging software (Olympus America Inc.). WGA-positive luminal signals were quantified using Imaris software (Bitplane).
3
Sphere Formation Assay for Tumor Cells
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After being treated with IL-6 signaling elicitors, including IL-6 and TNF-α/IL-6/sIL-6R (TNF-α: 20 ng/mL, IL-6: 5 ng/mL, and sIL-6R: 100 ng/mL), for two days, cells were trypsinized, suspended in a serum-free medium, and filtered through 40-μm Falcon strainers (BD Biosciences). Subsequently, a single-cell suspension was prepared (10 cells/mL), seeded in 96-well plates (100 μL per well), and cultured in a serum-free medium supplemented with 20 ng/mL of basic fibroblast growth factor (bFGF; Sigma-Aldrich), 20 ng/mL of epidermal growth factor (EGF; Sigma-Aldrich), and 20 µL/mL of B27 supplement (Invitrogen). Afterward, wells containing only one cell were labeled sequentially from 1 to 40 and observed daily, while wells without cells or containing more than one cell were not included in the analysis. Six days later, the diameters of the floating spheres were determined using DP2-BSW imaging software (Olympus). The cells were considered tumorigenic if spheres with a diameter of >40 μm were observed.
4
Evaluating Tumor Formation Potential
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After IL-6 signaling elicitor treatment, including IL-6 and TNF-α/IL-6/sIL-6R (TNF-α: 20 ng/mL, IL-6: 5 ng/mL, and sIL-6R: 100 ng/mL), for two days, cells were filtered through 40-μm Falcon strainers (BD Biosciences) and seeded into the upper layer of 0.3% (w/v) noble agar (Sigma-Aldrich) in six-well plates at a density of 2500 cells/well to evaluate their tumorigenicity [29 (link)]. Seven days later, the diameters of the tumor colonies (25 random 200× fields) were measured using DP2-BSW imaging software (Olympus, Tokyo, Japan). Because not all of the colonies were spherical, the maximal diameter of each colony was determined. Cells were considered tumorigenic based on the number of colonies with a diameter of >50 μm. Subsequently, the cells were photographed after another incubation period of seven days.
5
Histological Evaluation of Tick-Infested Rabbit Skin
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For histological evaluation, skin samples were collected from tick-infested rabbits (3 days post-infestation in histamine-treated rabbits and 6 days post-infestation in antihistamine-treated rabbits) and fixed in a 10% neutral buffered formalin solution. Afterward, fixed tissues were routinely processed [10 ], embedded in paraffin, and then sectioned at 5-μm thickness using a microtome at room temperature. Tissue sections were stained with hematoxylin and eosin (H & E) and examined under a microscope. Colored images were taken from H & E-stained skin sections using an Olympus BX51 microscope and Olympus DP72 camera equipped with DP2-BSW imaging software (Olympus, Tokyo, Japan).
6
Quantifying CD4 and CD8 Lymphocytes in CIN
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Serial section analysis for CD4 and CD8 was performed. Images were captured focusing on regions of interest (ROIs) using an OLYMPUS BX51 microscope and a DP72 OLYMPUS digital camera, using DP2-BSW imaging software (Olympus Corporation, Tokyo, Japan). ROIs were defined as: in normal epithelium, stroma immediately beneath normal epithelium, CIN 1/2/3 epithelium, and stroma immediately beneath CIN 1/2/3. Cytoplasmic immunoreactivity for CD4 and CD8 were semi-quantitated by consensus of two pathologists who were blinded to the clinicopathological data. Stained lymphocytes were quantitated by normalization against the area of the ROI (mm2) and given a score of either 0 (≤5 lymphocytes), 1+ (6-50 lymphocytes), 2+ (51-99 lymphocytes) or 3+ (>100 lymphocytes). Cells that were scored as 1+ or 2+ were considered as low rate of infiltration and those scored 3+ were considered as high rate of infiltration.
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