Tissue samples for sections were fixed overnight in 4% paraformaldehyde and embedded in paraffin wax. Antibodies and labelling procedures are described in the Supplementary Material . Image analysis was performed using a Leica TCS SP5 confocal microscope (fluorescence microscopy) or a Zeiss Axiophot microscope equipped with a Zeiss AxioCam HRc camera (haematoxylin and eosin and Herovici staining).
Tcs sp5 confocal microscope
Manufactured by Zeiss
Sourced in Germany
The TCS SP5 is a high-performance confocal microscope designed for advanced imaging applications. It features a modular architecture that allows for customization and integration of various detection systems. The TCS SP5 provides users with a versatile platform for performing detailed analysis of samples at the cellular and sub-cellular level.
Automatically generated - may contain errors
Lab products found in correlation
Axiocam, by Zeiss (4 mentions)
Axiocam hrc camera, by Zeiss (2 mentions)
Axiophot microscope, by Zeiss (2 mentions)
Axio imager a2, by Zeiss (2 mentions)
3 3 diaminobenzidine dab substrate solution, by ScyTek Laboratories (2 mentions)
Karyomax colcemid, by Thermo Fisher Scientific (2 mentions)
Dapi mounting medium, by Thermo Fisher Scientific (2 mentions)
Apotome 2, by Zeiss (2 mentions)
Dimerthylenastron dme, by Merck Group (2 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Lsm 880, by Zeiss (1 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
Airyscan module, by Zeiss (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Axio imager upright microscope, by Zeiss (1 mentions)
30 protocols using tcs sp5 confocal microscope
1
Tissue Preparation and Imaging
2
Mitotic Spindle Visualization Protocol
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Y235T cells were cultured in a 12-well plate with a 19 mm Ø round coverslip for at least 48 h without any treatment. Cells were extracted with microtubule stabilization buffer (MSB) for 5 min, followed by 10 min fixation with 4% PFA. In alternative experiments, cells were fixed for 3 min with ice-cold methanol without MBS treatment. Cells were stained with α-tubulin specific antibody to visualize spindles and with γ-tubulin specific antibody to visualize spindle poles, and with DAPI to visualize chromosomes. A total of 270–710 cells in mitosis from 3–4 independent experiments were counted. The pictures were acquired with the Zeiss TCS SP5 confocal microscope using a 63 × objective.
3
Immunofluorescence Staining of Cytoskeleton
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Cells were seeded and grown in a 12-well plate with a 19 mm Ø round coverslip in each well, and cultured for at least 48 h. For α-tubulin and γ-tubulin related staining, cells were extracted with microtubules stabilization buffer (MSB) for 5 min and then followed by 10 min fixation with 4% PFA or directly fixed in 3 min cold methanol without extraction. For F-actin related staining, cells were fixed with 4% PFA for 10 min. Then followed by two times wash with 1xPBS, 1% Triton X-100 in 1xPBS was used to permeabilized cells. After three times wash with 1xPBS, the cells were blocked with 1% BSA in 1xPBS at room temperature for one hour. Coverslips with cells were then incubated with primary antibody solution diluted in 1xPBS with 1% BSA over night at 4 °C. Next day, followed by three times wash in PBST (1xPBS containing 0,5% Tween), slides were incubated with an appropriate secondary antibody. Phalloidin reagent was mixed with secondary antibody and incubated with cells to visualize F-actin. Finally, samples were mounted with DAPI fluorescence mounting medium. Images were acquired with Zeiss TCS SP5 confocal microscope using a 63 × objective for colocalization and invadopodia detection. For the calculation of colocalization, Image J 1.53t (National Institute of Health, Bethesda, MD, USA) was used to measure Pearson’s coefficient value.
4
Immunohistochemical Analysis of Ureter and Bladder Samples
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Hematoxylin and eosin stainings, initial tests to establish IHC conditions, and the identification of the target proteins in human ureter and in the human and murine BC samples were carried out in our lab at the University of Ulm using 4 µm tissue sections in accordance with established protocols. IHC samples were stained with 3,3’-Diaminobenzidine (DAB) substrate solution (Scytek Laboratories, West Logan, USA). The pictures were acquired by using Zeiss TCS SP5 confocal microscope (Zeiss, Oberkochen, Germany) with 10 × and 63 × objectives. IHC experiments to evaluate ORP3 expression in 190 MIBC, as well as the in 26 normal human bladder samples were performed at the University Hospital Erlangen. An expert pathologist (Dr. M. Eckstein) stained the samples and assessed signal intensities using Axio Imager A2 (Zeiss, Oberkochen, Germany). The MIBC samples are from a previously described, well-characterized MIBC cohort [18 (link), 19 (link)] (see also Figure S2). Ethical approval for this study was obtained by the ethical review board of the Friedrich-Alexander-University Erlangen-Nürnberg (Erlangen, Germany; approval number: no. 3755 and 329_16B). All patients agreed and gave informed consent, and all analyzes were carried out in accordance with the Declaration of Helsinki.
5
Cell Migration Evaluation Assays
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Evaluation of cell migration by Wound healing assay and cell migration/invasion by Boyden chamber assay were performed as described previously [23 (link)]. Pictures were acquired using a 5× objective in case of the Wound healing assay and 10× objective in case of the Boyden chamber assay, using a Zeiss TCS SP5 confocal microscope.
6
Cell Migration Evaluation Assays
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Evaluation of cell migration by Wound healing assay and cell migration/invasion by Boyden chamber assay were performed as described previously [23 (link)]. Pictures were acquired using a 5× objective in case of the Wound healing assay and 10× objective in case of the Boyden chamber assay, using a Zeiss TCS SP5 confocal microscope.
7
Immunofluorescence Staining and Imaging Protocol
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Paraffin sections were de-paraffinized and treated with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0) for antigen retrieval (microwave). Blocking was performed with 1% BSA, 2.5% goat serum and 0.5% saponin in PBS overnight at 4°C. Primary antibodies were added 1:50 (ANX) or 1:1000 (H. pylori) (S3 Table ) in PBS/1% BSA/2.5% goat serum/0.5% saponin and samples were incubated overnight at 4°C. After washing, secondary antibodies (1:1000 in PBS/0.05% Tween-20/2.5% goat serum) were applied for 1 h at RT in the dark. Sections were stained with DAPI (5 μg/ml) for 10 min at RT in the dark and after washing were embedded in Fluorescence Mounting Medium (Dako). Sections were visualized using the Leica TCS SP5 confocal microscope for quantification of ANX signal or the confocal laser scanning microscope LSM880 (Zeiss) with Airyscan Module for co-localization studies. Imaris Bitmap 8.7 was used for co-localization analysis and Imaris software was used for the 3D reconstruction and rendering images.
8
Immunofluorescence Staining for Nuclear p50
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Cells were washed once with PBS and fixed using 4 % paraformaldehyde for 20 min. Then, cells were permeabilized with 0.3 % Triton X-100 in PBS for 5 min and saturated with blocking solution containing 5 % FBS and 0.3 % Triton X-100 in PBS for 30 min at RT. Primary antibodies CD11b (1:100), GFAP (1:100), and p105/p50 (1:100) diluted in blocking solution were incubated 1 h at RT. After several washes, the cells were incubated 1 h at RT with secondary antibodies Alexa-fluor 488 and Alexa-fluor 546 (1:200, Life Technologies), and after repeated washes, the cells were mounted using Mowiol reagent containing Hoechst (Roche). Images were acquired with a Leica TCS SP5 confocal microscope using Zeiss 63X objective. Quantifications of nuclear p50 were performed using ImageJ software. In detail, the mean of fluorescence intensity was calculated as p50 nuclear fluorescence intensity divided by the nuclear area and expressed as nuclear intensity/μm2. Quantification was performed in blind, and at least ninety cells were randomly chosen in four independent experiments per genotype.
9
Quantifying Spinal Cord Immunostaining
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Six thoracic spinal cord sections per animal from at least six animals per group were taken. Staining was quantified using the ImageJ software (NIH; Bethesda, MD, USA). Sections were analyzed by immunofluorescence on a Leica TCS SP5 confocal microscope and with a Zeiss Axiocam high-resolution digital color camera for IHC.
10
Actin Dynamics and Cell Invasion
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This assay was performed as described by Diaz [25 (link)]. Here, 3 × 104 RT4 or 1 × 104 T24 cells were seeded in 12-well plates with coverslips, coated with Gelatin-FITC. The cells were incubated in complete RPMI1640 medium at 37 °C in a humidified atmosphere with 5% CO2 for 3 days. The cells were then fixed with 4% PFA for 10 min, permeabilized with 1×PBS containing 0.1% Triton X-100 and blocked with 0.3% BSA for 30 min. After washing with 1×PBS, F-actin was visualized by staining with Alexa Flour-647 Phalloidin for 1 h at room temperature. Nuclei were stained with mounting medium containing DAPI. Confocal microscopy analyzes were performed using Zeiss TCS SP5 confocal microscope with a 40× objective. Degradation area was measured by using the ImageJ program, and the normalized area was calculated by the area dividing the cells in the whole picture.
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