Immunohistochemical staining was done on cells. At first, the slides were blocked with 5% normal serum, and then the primary antibodies were incubated with the slides overnight. After washing three times, slides were incubated with the secondary antibodies and ABC solution using the ABC kit (Vector Laboratories). Slides were then stained with a diaminobenzidine solution. Nuclei were stained by hematoxylin. The images from these slides were analyzed by NIS Element software (Nikon Instruments, Melville, NY) in the Nikon microscope image system (Nikon Instruments).
Nis element software
Manufactured by Nikon
Sourced in Japan, United States, Germany, France
NIS-Element software is a comprehensive imaging and analysis platform designed for a wide range of microscopy applications. It provides a suite of tools for image acquisition, processing, analysis, and documentation.
Automatically generated - may contain errors
Lab products found in correlation
Microscope image system, by Nikon (9 mentions)
Antigen unmasking solution, by Vector Laboratories (7 mentions)
Abc kit, by Vector Laboratories (6 mentions)
Eclipse ti microscope, by Nikon (6 mentions)
Eclipse ti, by Nikon (6 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (6 mentions)
Proteinase k, by Vector Laboratories (5 mentions)
Eclipse 80i, by Nikon (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
Eclipse ti e inverted microscope, by Nikon (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Orca flash 4.0 camera, by Hamamatsu Photonics (3 mentions)
Orca r2 camera, by Hamamatsu Photonics (3 mentions)
A1rsi, by Nikon (3 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (3 mentions)
Ti e inverted microscope, by Nikon (3 mentions)
A1r confocal microscope, by Nikon (3 mentions)
A1 laser scanning confocal microscope, by Nikon (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Fluo 4 am, by Thermo Fisher Scientific (3 mentions)
210 protocols using nis element software
1
Immunohistochemical Staining Protocol
2
Measuring Worm Length and Fixation
3
Fecal Egg Quantification and Worm Burden Analysis in Hamsters
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Faeces from individual hamsters from each group were collected 6 weeks after challenge, and eggs per gram feces (EPG) were counted and calculated using a modified formalin-ether acetate technique [26 (link)]. Briefly, 2 grams of hamster feces were fixed in 10 ml of 10% formalin solution, filtered through two layers of gauze, the filtered solution centrifuged at 500 g for 2 minutes, and the supernatant discarded. The pellet was re-suspended with 7 ml of 10% formalin, vigorously mixed with 3 ml ethyl acetate and then centrifuged at 500 g for 5 minutes. The pellet was dissolved in 1 ml of 10% formalin solution and examined in duplicate under light microscopy at 400x magnification. EPG was calculated as follows: (average of number eggs x total drops of fecal solution)/ g of feces. Whole livers from the hamsters were dissected in NSS and adult worms were collected and counted. The percent reduction of worm recovery was calculated as previously described [27 (link)]: % Worm reduction = ((Wc–We) / Wc) x 100 (Wc = worm burden in control group, We = worm burden in experiment group). To measure worm length, 21.4–42.2% of the total worms recovered from each group were randomly selected. Worms were washed three times with NSS and fixed in pre-warmed 10% formalin. Worms were photographed under microscopy and worm length was measured using NIS-Element software (Nikon, Japan).
4
Oxidative Stress Evaluation in Brain
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Oxidative stress markers were evaluated in brain areas homogenates. The proteins oxidation status was investigated using the OxyBlot Protein Oxidation Detection Kit (Merck Millipore, Burlington, MA, USA; Cat. N. S7150).35 ,36 (link) Briefly, 20 μg of proteins were separated in 10% by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to the nitrocellulose membrane. Transblotted membranes were incubated with specific primary and secondary antibodies diluted in 1% BSA in PBS-T. Positive bands were visualized by an enhanced chemiluminescence system (LiteAblot® Turbo, Cat. N. EMP012001; Euroclone) and the intensity of bands revealed by densitometry analysis with Image Lab software of ChemiDoc XRS (BioRad, Italy). In sections of brains monoclonal antibody specific for 8-oxo-2′- deoxyguanosine (8-oxo-dG), was used according to the manufacturer’s protocol (Table 2 ). After the incubation with the goat antimouse secondary antibody (Alexa Fluor 488), the sections were viewed using an Olympus BX51 Fluorescence Microscope Olympus Corporation, Europa SE & CO. KG, Germany). Percentage of area of immunoreaction was measured with the Nikon NIS Element software.
5
Immunohistochemistry of Kidney Tissues
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Immunohistochemistry was carried out on paraffin-embedded kidney sections [15 (link)]. Antigen unmasking solution (Vector Laboratories, Burlingame, CA, USA) was used to retrieve the antigen after fixation. Three percent H2O2 was used to block endogenous peroxidase activity before blocking. After incubation with 5% normal serum, slides were incubated with primary antibodies in a humidified chamber overnight. After washing, kidney sections were incubated with secondary antibodies, followed by ABC solution as previously reported [16 (link)]. Diaminobenzidine solution was applied to visualize the secondary antibody conjugated with horseradish peroxidase (HRP). Red substrate was used to visualize the secondary antibody conjugated with alkaline phosphatase (AP). Hematoxylin was used for nuclear staining. The sections were dehydrated with ethanol, cleared with histoclear, and mounted with mounting medium. NIS Element software (Nikon Instruments, Melville, NY, USA) with Nikon microscope image system (Nikon Instruments) were utilized to quantify the protein expression levels in the kidney.
6
Quantifying Mutant p53 in Cholangiocytes
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To investigate levels of p53 mutation [88 (link)] in cholangiocytes, paraffin-embedded tissue sections were deparaffinized and rehydrated by standard methods. Thereafter, sections were incubated with monoclonal mouse anti-p53 (mutant, clone Ab-3 PAb240 catalogue no. OP29-200UG) (Merck, Darmstadt, Germany) diluted 1:100 in PBS at 4°C overnight, and after thorough washing, probed with goat anti-mouse IgG-HRP (Invitrogen, Carlsbad, CA) diluted 1:1,000 in PBS for 60 min at 25°C. The peroxidase reaction was developed with 3,3’- DAB and sections counterstained with Mayer’s hematoxylin for 5 min. A human CCA cell line served as the positive control for mutant p53 positivity [89 ,90 (link)]. Images of high-power fields (400x magnification) of the biliary epithelium were captured in five non-overlapping fields of each of the right, middle, and left liver lobes using a Zeiss Axiocam fitted with a ICc5 camera and NIS-Element software (Nikon). The percentage of mutant p53-positive cholangiocytes was determined by scoring positive cells from 500 to 800 cholangiocytes from the right, middle, and left lobes of the liver using imageJ.
7
Time-lapse Imaging of Cell Migration
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Cells were seeded at a low density and kept at 37 °C under 5% CO2 in an incubator chamber for time–lapse video recording (Okolab). Cell movements were monitored with an inverted microscope (Eclipse Ti–E; Nikon Corporation, Tokyo, Japan) using a 10X/0.25 NA Plan objective lens.
Images were acquired every 10 min for a time–lapse of 10 h with a CCD video camera using NIS–Element software (Nikon). Image stacks were analyzed with ImageJ software, and at least 100 cells/condition were manually tracked using the MtrackJ plugin. We excluded dividing cells as well as cells that exited the imaged field during the time–lapse acquisition period. The mean speed parameter was considered for the data analyses. At least 6 fields for each condition were analyzed in each independent experiment. At least three independent experiments were performed for each experimental condition.
Images were acquired every 10 min for a time–lapse of 10 h with a CCD video camera using NIS–Element software (Nikon). Image stacks were analyzed with ImageJ software, and at least 100 cells/condition were manually tracked using the MtrackJ plugin. We excluded dividing cells as well as cells that exited the imaged field during the time–lapse acquisition period. The mean speed parameter was considered for the data analyses. At least 6 fields for each condition were analyzed in each independent experiment. At least three independent experiments were performed for each experimental condition.
8
Immunohistochemical Analysis of Kidney Samples
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Immunohistochemical staining was performed on paraffin sections as described36 ,37 (link). After antigen retrieval, endogenous peroxidase activity was quenched with 3% hydrogen peroxide. The kidney sections were incubated with primary antibodies, appropriate secondary antibodies and ABC solution sequentially as described9 (link). Immunoreactivity was detected with diaminobenzidine. Sections were counterstained with hematoxylin and mounted using histomount. A Nikon microscope image system (Nikon Instruments, Melville, NY) was used to capture the images and analyzed using a NIS Element software (Nikon Instruments, Melville, NY) in a blinded fashion by one observer4 (link),6 (link).
9
Immunofluorescence Analysis of Fracture Samples
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Harvested fracture samples were fixed in 10% formalin at 4°C for 24 h, decalcified in 14% EDTA (pH 7.4) for 2 weeks at 4°C with constant agitation, and processed in paraffin or frozen blocks. Immunofluorescence was performed using the following primary antibodies: anti-GFP (NB100-1770; Novus Biologicals), anti-Ki67 (14-5698-82; eBioscience), anti-RFP (600-901-379; Rockland Antibodies), anti–NF-κB (ab16502; Abcam), anti-TNF (ab34674), anti-F4/80 (ab6640), anti-CD4 (ab183685), anti-IKKβ (ab124957), anti-CD271 (ab52987), anti-Sca1 (ab51317), anti–inducible nitric oxide synthase (iNOS) (ab15323), anti-CD163 (ab182422), and anti–TGF-β1 (ab92486). The DeadEnd TUNEL System (G3250; Promega) or Annexin V (A13204; Invitrogen) was used to detect apoptotic cells. Percent-positive cells and mean fluorescence intensity (MFI) were quantified using NIS-Element software (Nikon).
10
Visualizing Microtubule Dynamics with TOG Domains
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