PH-PLCD1-GFP, PH-PLCD1-iRFP, and TfR-GFP plasmids were purchased from Addgene (plasmids 51407, 66841, and 45060, respectively) and were transfected into cultured HPAECs using the Neon Transfection System (Thermo Fisher) 2 d before imaging under optimized conditions (1,270 V, 20-ms pulse length, with two pulses). All of the images were captured using a confocal laser-scanning microscope (A1R HD25; Nikon) equipped with ×60 (ApoTIRF) and ×100 (SR ApoTIRF) objectives at the following excitation/emission wavelengths: EGFP-D4, PH-PLCD1-GFP, and TfR-GFP (488, 525/50 nm); mCherry-D4 (561, 595/50 nm); and PH-PLCD1-iRFP (640, 700/75 nm) using NIS-Elements C software (Nikon). The obtained images were merged using Adobe Photoshop software. Signals of D4-based sensors were quantitatively analyzed by Image Pro-10 software (Media Cybernetics).
Image pro 10
Manufactured by Media Cybernetics
Sourced in United States
Image-Pro 10 is a powerful image analysis software designed for a wide range of applications. It provides advanced tools for image processing, measurement, and analysis, enabling users to extract valuable insights from digital images. The software offers a comprehensive set of features to support various imaging workflows, catering to the needs of researchers, scientists, and professionals across diverse industries.
Automatically generated - may contain errors
Lab products found in correlation
Nis elements c software, by Nikon (1 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
A1r hd25, by Nikon (1 mentions)
Eclipse ti fluorescence microscope, by Nikon (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Citrisolv, by Avantor (1 mentions)
Bx51 fluorescence microscope, by Media Cybernetics (1 mentions)
Hemalaun, by Merck Group (1 mentions)
Roti histofix formaldehyde fixation reagent, by Carl Roth (1 mentions)
Dp74 color camera, by Olympus (1 mentions)
Eclipse 80i microscope, by Nikon (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Alexa conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Vectashield mounting media, by Vector Laboratories (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Jung rm2055, by Leica (1 mentions)
Zen blue 2, by Zeiss (1 mentions)
22 protocols using image pro 10
1
Intracellular Organelle Imaging in HPAECs
2
Quantifying Intestinal Goblet Cells
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Four sections of the small intestine from each rat were stained with Alcian Blue using a standard procedure, as previously described (Ducray et al. 2019 ). Enumeration of goblet cells was according the protocol proposed by Trevizan et al. (2016 ). Six images from each section were taken with a digital camera (Olympus BX50) coupled to an optical microscope with a 20x objective. The number of goblet cells present in a 0·96 mm2 in the mucosa of each animal were quantified using ImagePro 10 software (Media Cybernetics, Rockville, MD).
3
Skin Pore Analysis with VISIA-CR
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Facial images for skin pore analysis were obtained using VISIA‐CR (CANFIELD, Fairfield, CT, USA). Skin pore analysis was performed on the forehead, cheeks, and perioral areas using the cross‐mode from VISIA‐CR. Several filters in Image‐Pro 10 software (Media Cybernetics, Silver Spring, MD, USA) were used to emphasize the skin pores in the analysis area. The skin pore area (measured in pixels) was then analyzed.
4
Fluorescence Imaging of Intestinal Tissue
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The small intestines (duodenum, jejunum, and ileum) from sham and VSG control mice were paraffin-embedded and sectioned onto slides by the University of Michigan In-Vivo Animal Core. Paraffin was removed using Citrisolv (VWR). The slides were incubated overnight at 4°C with primary antibody (see above). The slides were stained with corresponding secondary antibodies conjugated to fluorescence dyes (see above) for 2 hours at room temperature. The slides were mounted in an antifade fluorescence mounting medium containing DAPI (VECTASHIELD with DAPI, Vector Laboratories, catalog H-1200). Fluorescence images were obtained using an Olympus IX73 inverted fluorescence microscopy system or Nikon Eclipse Ti fluorescence microscope with a motorized X-Y stage system. Images were analyzed using Olympus cellSens imaging software or Image Pro 10 software (Media Cybernetics).
5
Cerebellum Cytoarchitecture Evaluation in Hypoxia
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To evaluate the effect of hypoxia injury and GH treatment upon tissue cytoarchitecture, cerebellums were collected and fixed in Carnoy´s solution for 24 h at 4 °C, dehydrated in ethanol, and embedded in paraffin wax. Tissue sections (10 µm) were cut using a microtome and mounted onto charged slides [14 (link)]. For histological analysis, slides were stained with hematoxylin-eosin (H-E) [57 (link)]. Cerebellum cell-layer thickness was determined in slices stained with H-E in at least 10 microscopic fields per animal, and 3 individual animals per experimental group were examined. Morphometrical analysis was performed in the same area of the folia for equivalent group comparisons. Images were captured using an Olympus BX51 fluorescence microscope (Tokyo, Japan) and analyzed with Image Pro 10 software (Media Cybernetics, Rockville, MD, USA).
6
Analyzing Skin Pore Characteristics
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Facial images for skin pore analysis were taken using VISIA‐CR (CANFIELD, Fairfield, USA). Skin pore analysis was performed on the forehead, cheeks, and perioral area using the cross mode from VISIA‐CR. Several filters in Image‐Pro 10 software (Media Cybernetics, Silver Spring, USA) were used to emphasize the skin pores in the analysis area. The skin pore area (measured in pixels) was analyzed.
7
Immunohistochemical Analysis of Protein Expression
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T-47D, MCF-7 and BT-20 cells were fixed subsequent to experimental treatment using ROTI® Histofix formaldehyde fixation reagent (4% formaldehyde in PBS, pH 7, Carl Roth, Karlsruhe, Germany). An indirect immunoperoxidase technique was applied to visualize the target protein expression. Antigen retrieval was performed in a 10 mmol/L sodium citrate buffer (pH 6.0) followed by inhibition of endogenous peroxidase by incubation for 30 min with 3% H2O2. After two washes in trissaline buffer, slides were incubated with 1% goat serum for 30 min to block unspecific staining. Following overnight incubation with the target protein antibody (Supplementary Table 3) at RT and two washing steps with PBS, slides were exposed to ImmPRESS peroxidase polymer reagent (BIOZOL, Eching, Germany) for 60 min at RT. Staining was achieved by 3,3-diaminobenzidine (DAB; BIOZOL, Eching, Germany) and the cells were counterstained with hemalaun (Merck). The colour intensity was quantified and evaluated with the software Image-Pro® 10 (MEDIA Cybernetics, Rockville, USA), according to the manufacturer’s specifications. For better comparison, the percentage staining in comparison to the cell surface was calculated. A staining below 5 % of the area was considered negative.
8
Quantifying Vascular Media Thickness
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Formalin fixed and paraffin embedded (FFPE) tissue was immunofluorescence stained for α-smooth muscle actin-stained vascular vessels using the protocol outlined in Table 1
9
Zika Virus Infection Characterization in Vero E6 Cells
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Vero E6 cells were infected with ZIKV at MOI=0.05 and incubated at 37°Cfor 72 h. Cells were washed thrice with ice-cold PBS, fixed in 4% paraformaldehyde for 10 min, and permeabilized in 0.2% Triton X-100 for 10 min. The cells were blocked with 1% bovine serum albumin (BSA) for 1 h. Cells were incubated with ZIKV anti-Envelop antibody (1:1,000) (Genetex, USA) at room temperature for 2 h and washed three times with PBS (5 min for each wash). The cells incubated with Alexa-conjugated secondary antibodies (Invitrogen) (1:1,000) for 1 h at room temperature, after which the cells were washed three times with PBS. The cells were then stained with DAPI and mounted using Mounting Medium. Fluorescence images were visualized using the Zeiss LSM 710 confocal microscope (Oberkochen, Germany). Calculation of green immunofluorescence intensity was done using the Image pro 10 (Media cybernetics, USA) Imaging and Processing Analysis Software with signaling intensity normalized to loading control.
10
Immunofluorescence Analysis of Tight Junction Proteins
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Histological sections were deparaffinized in Hemo‐Di x 2 changes for 5 min, hydrated with 100, 95 and 70% ETOH and distilled water. Sections were blocked for 1 h in Odyssey blocking buffer (LiCor, Lincoln, CA) and incubated for 2 h at 4°C with primary antibodies against claudin, occludin, ZO‐1 and JAM‐A proteins. Sections were washed with PBS and incubated with secondary antibodies (1 : 100 dilution) for 1 h, then washed with PBS/0·01% Tween. Sections were mounted with VectaShield Mounting Media (Vector Laboratories, Burlingame, CA). Fluorescence images from each section were obtained with a digital camera (Olympus BX50) coupled to an optical microscope. The images were analysed with Image‐Pro10 (Media Cybernetics). Three fields per each of three sections from each rat were analysed.
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