To visualize the localization of bacteria carrying the malX gene, which encodes for maltodextrin-degrading enzymes,14 (link), 38 (link), 39 (link) ileal tissues were fixed in methanol-based Carnoy’s fixative (60% absolute methanol, 30% glacial acetic acid, 10% chloroform), embedded in paraffin blocks, and sectioned. Five-µm sections were deparaffinized and hybridized with 250-ng E. coli-Cy3 probe (5’-Cy3-CAT CTT CAC AGC GAG TTC-3’), 500-ng MalX-Alexa488 probe (5’Alex488-ACG CGT TTC CTT TCG CAA-Alexa488-3’), and Eubacteria338-Alexa647 probe (5’Alexa647-GCT GCC TCC CGT AGG AGT-3’) or buffer-only controls in 20-mM Tris-HCL, 0.01% SDS, 0.9M NaCl at 46°C overnight.40 (link) Slides were then rinsed twice with water, incubated 5 minutes in 20-mM Tris-HCL, 0.9M NaCl at 46°C, rinsed twice with water, dried 10 minutes at 46°C, and applied with Vectashield containing DAPI (Vector Labs) and coverslips. Imaging was acquired using a Leica TCS-SP spectral laser scanning confocal microscope equipped with a Q-Imaging Retiga EXi cooled CCD camera and Image ProPlus Capture and Analysis software (Media Cybernetics). Image z-stacks were collected every 0.49 μm spanning the full thickness of cells and exported for image analysis using Image ProPlus Capture and Analysis software (Media Cybernetics).
Imagepro plus capture and analysis software
Manufactured by Media Cybernetics
Sourced in Germany
ImagePro Plus is a powerful software suite for image capture, analysis, and processing. It provides a comprehensive set of tools for users to acquire, enhance, measure, and interpret digital images from a variety of sources. The software offers a user-friendly interface and advanced features to support a wide range of scientific and research applications.
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Lab products found in correlation
Oct medium, by Sakura Finetek (5 mentions)
Fluorescent or bright field imaging microscopy, by Leica (4 mentions)
Hematoxylin, by Vector Laboratories (2 mentions)
Rabbit anti vimentin, by Cell Signaling Technology (2 mentions)
Rabbit anti fibronectin, by Merck Group (2 mentions)
Rabbit anti n cadherin, by Thermo Fisher Scientific (2 mentions)
Optimal cutting temperature medium, by Sakura Finetek (1 mentions)
Rat anti f4 80, by Bio-Rad (1 mentions)
Anti gr 1, by Bio-Rad (1 mentions)
Rat anti f4 80, by Thermo Fisher Scientific (1 mentions)
Rat anti mouse cd206, by Bio-Rad (1 mentions)
Vectashield containing dapi, by Vector Laboratories (1 mentions)
7 protocols using imagepro plus capture and analysis software
1
Localization of Maltodextrin-Degrading Bacteria
2
Immunofluorescence Quantification of SerpinB2 in Tissue Sections
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Tissues were collected at the indicated times, snap-frozen in optimal cutting temperature (OCT) medium (Sakura Finetek, Japan), and 8-μm sections were prepared and stained with the following antibodies; rabbit anti-SerpinB2 (1:100) (Abcam), as described previously11 (link),15 (link),38 (link). Stained sections were analyzed using fluorescent or bright-field imaging microscopy (Leica) and ImagePro Plus Capture and Analysis software (Media Cybernetics). SerpinB2 positive areas were quantified in 15 independent fields/section using Image Pro-Plus software11 (link),38 (link).
3
Immunofluorescence Analysis of Signaling Pathways in Breast Cancer
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BC tumor tissues were collected at the indicated times, snap-frozen in optimal cutting temperature medium (Sakura Finetek), and 8-μm sections were prepared and stained with the following antibodies: rabbit anti pAKT-S473 (1:400), pSMAD3(1:200), pERK1/2 (1:200), AKT (1:400), SMAD3 (1:500), and ERK1/2 (1:400), as described previously22 (link),39 (link),42 (link),43 (link). Stained sections were analyzed using fluorescence imaging microscopy (Leica) and ImagePro Plus Capture and Analysis software (Media Cybernetics). Positive areas were quantified in 15 independent fields/section using the Image Pro-Plus software22 (link),39 (link),42 (link),43 (link).
4
Immunohistochemical Analysis of WAVE3 and β-Catenin in Tumor and Lung Tissues
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Tumors and lung tissues were harvested and snap-frozen in optimal cutting temperature (OCT) medium (Sakura Finetek; Torrance, CA), and 8-μm sections were prepared. Tumor sections were stained with the following antibodies: rabbit anti-WAVE3 and mouse anti-β-Catenin, followed by Alexa 488 anti-mouse or Alexa 568-conjugated goat anti-rabbit IgG. Lung sections were stained with hematoxylin (Vector Laboratories) and eosin (Fisher Scientific Co.; Kalamazoo, MI) following the manufacturer’s protocol. Confocal or bright-field imaging microscopy (Leica, Wetzlar, Germany) was used to capture the images and ImagePro Plus Capture and Analysis software (Media Cybernetics; Rockville, MD) was used to analyze the stained sections from 10 to 15 independent fields/sections.
5
Immunohistochemical Analysis of Tissue Markers
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Tissues were collected at the designated times and snap-frozen in optimal cutting temperature (OCT) medium (Sakura Finetek, Japan), and 8-μm sections were prepared and stained with the following antibodies; Rabbit anti-fibronectin (1:100) (Millipore, Temecula, CA, USA), rabbit anti-vimentin (1:100) (Cell Signalling Technology, Danvers, MA, USA), rabbit anti-N-cadherin (1:1000) (Thermo Scientific, Rockford, IL, USA), as described previously13 (link),41 (link). Stained sections were analysed using fluorescent or bright-field imaging microscopy (Leica, Germany) and ImagePro Plus Capture and Analysis software (Media Cybernetics, Rockville, MD). Vimentin, Fibronectin and N-Cadherin positive areas were quantified in 15 independent fields/section using Image Pro-Plus software41 (link),44 (link).
6
Quantifying Macrophage Subsets in Tissue Sections
7
Immunohistochemical Analysis of Tumor Tissues
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Tumour and lung tissues were harvested and snap-frozen in optimal cutting temperature (OCT) medium (Sakura Finetek, Torrance, CA, USA), and 8-μm sections were prepared. Tumour sections were stained with the following antibodies: rabbit anti-fibronectin (Millipore, Temecula, CA, USA), rabbit anti-vimentin (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-N-cadherin (Thermo Scientific, Rockford, IL, USA), followed by Alexa 488 or Alexa 568-conjugated goat anti-rabbit IgG. Lung sections were stained with hematoxylin (Vector Laboratories, Burlingame, CA, USA) and eosin (Fisher Scientific Co., Kalamazoo, MI, USA) following the manufacturer’s protocol. We used fluorescent or bright-field imaging microscopy (Leica, Wetzlar, Germany) and ImagePro Plus Capture and Analysis software (Media Cybernetics, Rockville, MD, USA) to analyse the stained sections. We used Image Pro-Plus software to quantify stained areas from 10–15 independent fields/sections.
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