Tissue samples were fixed for in 4% paraformaldehyde for 16 h at 4 °C. Xylene and a descending ethyl alcohol series were used for deparaffinization and rehydration of 2.5 µM sections from formalin-fixed, paraffin-embedded tumor tissue. Next, tissues were stained with Mayer’s hematoxylin solution (Hospital Pharmacy, Klinikum rechts der Isar, Munich, Germany) for 5 min., followed by successive washes in tap water, deionized water and 95% acid ethanol for 5 min., 2 min. and 20 sec., respectively. Then sections were stained in alcoholic Eosin B (Hospital Pharmacy) for 2 min., followed by dehydration with an ascending alcohol series and mounting with Entellan NEW (Merck Chemicals GmbH, Darmstadt, Germany). Slides were examined using a Zeiss Axio Vert.A1 microscope and images were obtained using Zen Lite 2012 software (Carl Zeiss).
Zen lite 2012
Manufactured by Zeiss
Sourced in Germany
ZEN lite 2012 is a software platform for microscope control and image acquisition developed by Zeiss. It provides a user-friendly interface for managing and optimizing microscope settings, as well as tools for capturing, processing, and analyzing microscopy data.
Automatically generated - may contain errors
Lab products found in correlation
Axiocam erc5s camera, by Zeiss (3 mentions)
Primo star iled microscope, by Zeiss (2 mentions)
Axioscan, by Zeiss (2 mentions)
Anti fapα, by R&D Systems (2 mentions)
Anti fapα f11 24, by Thermo Fisher Scientific (2 mentions)
Anti mouse igg2a tritc, by Southern Biotech (2 mentions)
Anti mouse igg2b cy5, by Southern Biotech (2 mentions)
Goat anti fitc alexa 488 antibody, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse igg1 fitc, by Southern Biotech (2 mentions)
Lsm 510 confocal microscope, by Zeiss (2 mentions)
Immpact dab peroxidase hrp substrate, by Vector Laboratories (2 mentions)
Anti pdpn nz 1, by Thermo Fisher Scientific (2 mentions)
Thy 1a1, by R&D Systems (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Lsm 7 duo confocal microscope, by Zeiss (2 mentions)
Entellan new, by Merck Group (1 mentions)
Axio vert a1 microscope, by Zeiss (1 mentions)
Plasmin, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
42 protocols using zen lite 2012
1
Histological Analysis of Formalin-Fixed Tumor Tissue
2
Rapid Polyhydroxyalkanoates Imaging in Mgryph Cells
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Mgryph cells (100 μL) were taken from FSM cultures and stained with 5 μL of 0.1 mg mL−1 of pyrromethene-546 (Pyr-546) in dimethyl sulfoxide for polyhydroxyalkanoates (PHAs) imaging. A Zeiss Primo Star iLed microscope (Carl Zeiss Ltd., Cambridge, UK) fitted with a Zeiss AxioCam ERc 5s camera was used. Images were acquired within 1 min of fluorophore incubation and processed with the aid of Zeiss ZEN Lite 2012 software in auto exposure mode. Samples were excited with a Zeiss Led 470 nm light source and a 515 LP filter was employed for detection of Pyr-546 fluorescence.
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Mgryph cells (100 μL) were taken from FSM cultures and stained with 5 μL of 0.1 mg mL−1 of pyrromethene-546 (Pyr-546) in dimethyl sulfoxide for polyhydroxyalkanoates (PHAs) imaging. A Zeiss Primo Star iLed microscope (Carl Zeiss Ltd., Cambridge, UK) fitted with a Zeiss AxioCam ERc 5s camera was used. Images were acquired within 1 min of fluorophore incubation and processed with the aid of Zeiss ZEN Lite 2012 software in auto exposure mode. Samples were excited with a Zeiss Led 470 nm light source and a 515 LP filter was employed for detection of Pyr-546 fluorescence.
3
Visualizing PN-1 and Plasmin Interactions
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Primary human vSMC cultures were grown in 48-well plates or 8-well chamber slides and incubated with PN-1 alone (125 nM, in house), plasmin alone (25 nM, American diagnostic inc, Connecticut, USA) or PN-1 and plasmin together which were pre-incubated for 1 h at 37°C to allow complex formation, without or with RAP (Receptor associated protein) (50 μg/ml, Molecular innovation, Michigan, USA) and added to vSMCs for 2 h at 37°C.
First, cells were washed with PBS and fixed in PBS containing 3.7% paraformaldehyde, then permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS. Cells were blocked with 5% BSA in PBS and incubated with the following primary antibodies: rabbit anti-human PN-1 (1:600, in house), mouse anti-LRP-1 (1:250, Sigma-Aldrich), and mouse anti-plasmin/plasminogen (1:50, Thermo Scientific); this latter was used to detect both plasmin alone and plasmin-PN-1 complexes. Alexa Fluor®488 and 555-labeled (Life Technologies) secondary antibodies were used for visualization. Finally cells were washed and incubated with DAPI (4, 6-diamidino-2-phenylindole) to stain the nuclei. Staining was visualized using a Leica fluorescence microscope or a Zeiss LSM 780 confocal microscope and images were analyzed with the Zen Lite 2012 software (France).
First, cells were washed with PBS and fixed in PBS containing 3.7% paraformaldehyde, then permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS. Cells were blocked with 5% BSA in PBS and incubated with the following primary antibodies: rabbit anti-human PN-1 (1:600, in house), mouse anti-LRP-1 (1:250, Sigma-Aldrich), and mouse anti-plasmin/plasminogen (1:50, Thermo Scientific); this latter was used to detect both plasmin alone and plasmin-PN-1 complexes. Alexa Fluor®488 and 555-labeled (Life Technologies) secondary antibodies were used for visualization. Finally cells were washed and incubated with DAPI (4, 6-diamidino-2-phenylindole) to stain the nuclei. Staining was visualized using a Leica fluorescence microscope or a Zeiss LSM 780 confocal microscope and images were analyzed with the Zen Lite 2012 software (France).
4
Picrosirius Red Collagen Quantification
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Paraffin sections were dewaxed, rehydrated, and stained with picrosirius red solution (0.1% sirius red in saturated picric acid) for 1 h. Specimens were then washed with acidified water (0.5% acetic acid, v/v), dehydrated, and then mounted on coverslips. Assays were evaluated under polarized light. Fibers illuminated in green, yellow, and red were considered to be collagens and the color intensity of these fibers was measured on a gray scale using Zen lite 2012 software (Zeiss). Collagen content in the myointimal and adventitial layers was expressed as an intensity (arbitrary units), normalized to the area of interest.
5
Quantification of Collagen in Skin Tissue
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Skin was harvested and paraffin embedded for histological evaluation of the collagen dermal area by Masson’s trichrome staining (Sigma-Aldrich, St. Louis, USA). Masson’s stained full thickness skin sections were visualized on a Zeiss A1 microscope, and photographed and digitalized using an AxioCam ERc 5S camera and ZEN lite 2012 software (Zeiss, Jena, Germany). Collagen blue-stained fractional area was quantified using ImageJ software (http://rsb.info.nih.gov/ij) .
6
Synovial Tissue Immunohistochemistry and Immunofluorescence Protocols
7
Immunohistochemical Detection of ANO4
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Eyes of C57BL/6 J or Ano4KOCfa were fixated in Davidson fixative and embedded in paraffin. 5 µm sections were deparaffinized and antigen retrieval was performed by Proteinase K incubation. After blocking the tissue with 5% BSA, the sections were incubated with an antibody against Ano4 (1:200) overnight at 4 °C. Goat anti rabbit Texas Red (Invitrogen) was used as secondary antibody. Nuclei were counterstained using DAPI (3 µM, Invitrogen). Sections were imaged with an Axio Imager M2 fluorescence microscope (Zeiss,) and data was processed by ZEN lite 2012 Software (Zeiss).
8
Leaf Surface Microscopy Protocol
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The surfaces of leaves were examined by using a Zeiss Scope.A1 microscope equipped with the AxioCam MRc 5 camera and ZEN lite 2012 software (Carl Zeiss MicroImaging GmbH, Jena, Germany). The samples preparation was done by following the next steps: peel the epidermis from the backside of the leaf, mount the sample on a glass slide in distilled water, fix with a coverslip, and observe under the microscope at 40× magnification. For scanning electron microscopy (SEM), leaves were mounted on stubs using carbon double-sided adhesive tape without any treatment. The samples were examined and photographed using LYRA3 scanning electron microscope (LYRA3 XMU, Tescan, Brno, CzechRepublic) with Low Vacuum Secondary Electron Tescan Detector (LVSTD), at 15 kV and magnification 800×.
9
Cell Invasion Imaging and Analysis
10
Synovial Tissue Immunohistochemistry and Immunofluorescence Protocols
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Synovial tissue samples were frozen in Tissue-Tek OCT medium (Miles, Elkhart, IN) or formalin fixed and paraffin embedded (FFPE).
For immunohistochemistry, antigen retrieval was performed at pH 9 on FFPE sections using Tris-EDTA, 0.05% Tween 20 (10mM Tris Base, 1mM EDTA Solution, 0.05% Tween 20). Sections were stained using anti-FAPα (R&D) and anti-goat Horseradish peroxidase (HRP) (Dako). HRP staining was developed using the ImmPACT DAB Peroxidase HRP Substrate (Vector Labs). Images were acquired using the Zeiss Axio Scan and analysed with Zen lite 2012 software (Zeiss). Number of pixels was quantified and divided by a manually defined tissue area and the average number of pixels per unit area (pixel/UA) was calculated.
For immunofluorescence, acetone fixed frozen sections were incubated with anti-FAPα (F11-24, eBioscience), anti-PDPN (NZ-1.3, eBioscience) and anti-THY1 (Thy-1A1, R&D). These were detected with goat anti-mouse IgG1 FITC, anti-mouse IgG2a TRITC and anti-mouse IgG2b Cy5 (all Southern Biotech). To increase signal from FITC-channel, goat anti-FITC Alexa-488 antibody (Invitrogen) was used. Images were acquired using a Zeiss LSM 510 confocal microscope and ZEN pro 2011 imaging software.
For immunohistochemistry, antigen retrieval was performed at pH 9 on FFPE sections using Tris-EDTA, 0.05% Tween 20 (10mM Tris Base, 1mM EDTA Solution, 0.05% Tween 20). Sections were stained using anti-FAPα (R&D) and anti-goat Horseradish peroxidase (HRP) (Dako). HRP staining was developed using the ImmPACT DAB Peroxidase HRP Substrate (Vector Labs). Images were acquired using the Zeiss Axio Scan and analysed with Zen lite 2012 software (Zeiss). Number of pixels was quantified and divided by a manually defined tissue area and the average number of pixels per unit area (pixel/UA) was calculated.
For immunofluorescence, acetone fixed frozen sections were incubated with anti-FAPα (F11-24, eBioscience), anti-PDPN (NZ-1.3, eBioscience) and anti-THY1 (Thy-1A1, R&D). These were detected with goat anti-mouse IgG1 FITC, anti-mouse IgG2a TRITC and anti-mouse IgG2b Cy5 (all Southern Biotech). To increase signal from FITC-channel, goat anti-FITC Alexa-488 antibody (Invitrogen) was used. Images were acquired using a Zeiss LSM 510 confocal microscope and ZEN pro 2011 imaging software.
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