PreB/pCAGS or preB/FL-RAGE cells were seeded and cultured for the indicated hours before being photographed at 20× magnification in phase contrast with an Apotome microscope (Zeiss, Germany). Aggregates area of 3 different fields was measured with Axiovision Software™ Rel 4.7 (Zeiss). For “mixed” aggregation assays, a double colored assay was performed as already described with same modifications [37] (link), [38] (link). Briefly, preB cells or preB/FL-RAGE (that express GFP) were labelled fluorescent red with 5 µM of CellTracker™ Orange CMTMR (5-)and-6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine (Invitrogen) in RPMI 1640 medium for 45 min at 37°C. After extensive washing, cell lines were cultured as single cell suspensions or mixed in equal number (5×104) as indicated and allowed to aggregate in growth medium for 24 h at 37°C under 5% CO2. Images were taken using an Apotome microscope (Zeiss, Germany) with a 20× objective and Axiovision Software™ Rel 4.7 (Zeiss).
Axiovision software rel 4
Manufactured by Zeiss
Sourced in Germany
Axiovision Software™ Rel 4.7 is an imaging and analysis software developed by Zeiss. It is designed to provide an intuitive and comprehensive interface for acquiring, processing, and analyzing digital images from various microscopy techniques.
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Lab products found in correlation
Apotome microscope, by Zeiss (2 mentions)
Axioskop 2 microscope, by Zeiss (2 mentions)
Celltracker orange cmtmr 5 and 6 4 chloromethyl benzoyl amino tetramethylrhodamine, by Thermo Fisher Scientific (1 mentions)
Axio observer z1, by Zeiss (1 mentions)
Axioskop microscope, by Zeiss (1 mentions)
Calcofluor white, by Zeiss (1 mentions)
Eclipse 50i, by Nikon (1 mentions)
Dxm1200c, by Nikon (1 mentions)
Tcs sp8 x confocal microscope, by Leica (1 mentions)
Application suite af software, by Leica (1 mentions)
Application suite x las x software, by Leica (1 mentions)
Axiocam mrm digital camera, by Zeiss (1 mentions)
Axioplan, by Zeiss (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Donkey serum, by Merck Group (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Dako fluorescent mounting medium, by Agilent Technologies (1 mentions)
Dm irb, by Leica (1 mentions)
Fibronectin, by Merck Group (1 mentions)
11 protocols using axiovision software rel 4
1
Cell Aggregate Formation Assay
2
Quantifying DCX Immunoreactivity in RMS
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DCX immunoreactivity in the RMS was determined using ImageJ (version 1.43u, National Institutes of Health, Bethesda, MD, USA), in images acquired in an inverted microscope (Axio Observer Z1, Zeiss, Jena, Germany), using the Mosaix module from the AxioVision software Rel. 4.8.2 (Zeiss, Jena, Germany), in order to capture the entire RMS on each section. DCX-positive area was measured for each image in 3 boxes of 250 μm × 250 μm, randomly placed along the length of the RMS, a method similar to what was previously described (Kuhn et al., 1997 (link)).
3
Microscopic Analysis of Regenerating Protoplasts
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After covering the loaded microvessels with a coverslip, they were examined under an Axioskop microscope (Zeiss, Jena, Germany), equipped with a 32x long distance objective (Zeiss Neofluar, Jena, Germany), and a digital CCD camera (AxioCam MRm). The Calcofluor White signal was recorded through the filter set 49 (excitation at 365 nm, beamsplitter at 395 nm, and emission at 445 nm; Zeiss, Jena, Germany). Images were processed and analyzed using the AxioVision software (Rel. 4.8.2) (Zeiss, Jena, Germany). For the viability assays, between 100–250 cells were observed per data point. Between 181–350 cells were scored per data point for testing the geometry sensing of regenerating protoplast, and the loss of geometry sensing by inhibition of auxin efflux (Fig. 2 ). For the auxin-gradient analysis (Fig. 4 ) and cell geometry alterations by auxin gradient 62–186 cells were counted per area and time point, giving a total population of 408–688 individual cells per time point.
4
Microscopic Film Thickness Measurement
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To measure the thickness of the films, we adapted the Swiss Roll technique [21 (link)], originally developed to prepare tissues for paraffin or methacrylate embedding for histological analyses followed by optical microscopy (Nikon Eclipse 50i; Nikon, Tokyo, Japan). Films (n = 3) were observed using a light microscope with camera (Nikon digital camera, DXM1200C; Nikon, Tokyo, Japan). Film thickness was estimated processing the pictures with the Axiovision software Rel 4.8 (Carl Zeiss Microscopy GmbH, Germany).
5
Oligodendrocyte Dystroglycan Imaging Protocol
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Oligodendrocyte α- and β-dystroglycan images were obtained using a zeiss axiocam MRM digital camera and Zeiss Axioplan inverted epifluorescence microscope using 20× and 40× objectives, operating under the control of Axiovision software (Rel. 4.8; Carl Zeiss, Oberkocken, Germany). Oligodendrocyte dystrophin and MBP images were obtained using a Leica SP5 confocal microscope using 20× and 40× objectives, operating under Leica Application Suite AF software. The oligodendrocyte utrophin and all brain section images were obtained using a Leica TCS SP8 X Confocal Microscope using 20×, 40×, and 63× oil immersion objections, operating under Leica Application Suite X (LAS X) software.
6
Quantification of Aortic Calcification
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Mice thoracic aortas or aortic rings were fixed in 10% formalin and embedded in paraffin. A total of 5 to 7 μm sections were de-paraffinized and incubated with 1% silver nitrate solution under ultraviolet light for 20–40 min. After rinsing the specimens with several changes of distilled H2O, the unreacted silver was removed with 5% sodium thiosulfate for 5 min at room temperature. Then, the sections were counterstained with haematoxylin for 30 s and eosin for 3 min. The quantification of the Von Kossa positive area was made by taking images with an Axioskop II microscope (Zeiss, Oberkochen, Germany) using a digital camera (AxioCam Color, Zeiss). The entire aorta cross section was analysed with Axiovision Software Rel 4.7 (Zeiss), and the percentage of calcium content was defined as the Von Kossa positive area divided by the total area (μm2).
7
Immunofluorescence Analysis of RAGE Expression
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For IF analysis, HEK-pcDNA, HEK/FL-RAGE cells or a mix of equal number of both cell types (1×105 total number) were seeded on glass coverslips and after two days of culturing, fixed in 4% paraformaldehyde at room temperature (RT) for 30 min. Cells were then washed by PBS followed with 1 h blocking in PBS+5% donkey serum (Sigma). After washing 3 times with 1% donkey serum plus 0.1% Triton (DST), cells were incubated for 1 h at rt with goat anti-human RAGE antibody (4 µg/ml; cat. AF1145, R&D Systems) in DST buffer. Cells were then incubated for 1 h at rt with donkey anti-goat conjugated with Alexa Fluor 546 (1∶1000; Invitrogen, USA). Nuclei were couterstained with Hoechst 33342 (1∶2000; Invitrogen) for 10 min at RT. Coverslips were mounted with Dako Fluorescent mounting medium (DAKO, CA, USA). Images were taken using an Apotome microscope (Zeiss, Germany) with a 20× objective and Axiovision Software™ Rel 4.7 (Zeiss). A similar analysis was performed on R3/1-pLXSN or R3/1-FL-RAGE cells avoiding addition of 0.1% Triton during washes.
8
Cell Adhesion and Spreading Assay
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The assays were performed as described previously with following modifications [17] (link). Tissue culture plates were coated for 2 hours at 37°C with 10 µg/ml collagen I (C7661), laminin (L2020), fibronectin (all from Sigma, St. Louis, MO, USA) or PBS. For cell adhesion assay, after saturation of wells with DMEM +1% BSA, R3/1-pLXSN or R3/1-FL-RAGE cells (5×104) were resuspended in DMEM +10% FCS and seeded on coated 96-well plates in triplicate for 15 or 45 minutes at 37°C under 5% CO2. Cells were extensively washed with PBS, fixed with 4% paraformaldehyde, stained with crystal violet for 10 min, lysed with 2% SDS and read on an ELISA plate reader at 550 nm. For cell spreading assay, R3/1-pLXSN or R3/1-FL-RAGE cells (5×104) were resuspended in DMEM +10% FCS and seeded on 12-well coated plates in triplicate for 90 minutes at 37°C under 5% CO2. Cells were then washed with PBS and fixed with 4% paraformaldehyde. Photographs were taken at 40× magnification in phase contrast (Leica DM IRB). Cell surface area (µm2, 25–50 cells) was quantified using Axiovision Software™ Rel 4.7 (Zeiss).
9
Immunohistochemical Analysis of Melanoma
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The handling of patient material was performed according to the Göttingen ethics committee votum No. 13/5/17 and according to the Statement of the National Ethics Council on Biobanks for Research, Berlin, Germany. Human melanoma samples were collected only from patients who signed informed consent. Samples were pseudo‐anonymized, and immunohistochemical staining was performed. Briefly, 5‐μm slides of paraffin‐embedded normal skin, primary melanoma, and metastasis samples were deparaffinated and treated with target retrieval solution (Dako #S1699) for 20 min. After cooling, endogenous peroxidases were blocked with 3% H2O2, washed with PBS three times, and stained with primary antibody overnight in a wet chamber at 4°C (specifics and dilutions are listed in Appendix Table S7 ). Samples were afterward washed with PBS three times. Secondary antibody (goat‐anti‐mouse Vector #BA‐9200, dilution 1:150; rabbit‐anti‐goat Vector #BA‐5000, dilution 1:150; goat‐anti‐rabbit Vector #BA‐1000, dilution 1:150) was incubated and washed exactly as the primary antibody. Samples were blocked with streptavidin peroxidase (Calbiochem #189733). Photographs were taken with an Axio Imager M1 and recorded using the AxioVision software Rel 4.7 (Zeiss, Göttingen, Germany). The slight difference in staining color (brown‐red for TMX1 and deep red for NFAT1) is due to different secondary antibodies.
10
Immunohistochemical Quantification of IL6 in Mouse Aorta
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Mouse distal thoracic aortas were fixed in 10% formalin and paraffin embedded. Six micrometer sections were boiled for 20 min in Dako Target Retrieval Solution Citrate pH 9 (Aligent Technologies, Santa Clara, CA, USA) and then blocked in 5% goat serum in PBS-T for 45 min at room temperature. Primary antibody against IL6 (5 μg/mL, AF-406-NA, R&D Systems) was dissolved in 1% goat serum PBS-T and incubated overnight at 4 °C in a humidified chamber. Sections were counterstained with hematoxylin. Images were acquired with an Axioskop II microscope (Zeiss, Oberkochen, Germany) using a digital camera (AxioCam Color, Zeiss).
The quantification of IL6 signal was carried after acquiring the images with an Axioskop II microscope (Zeiss) using a digital camera (AxioCam Color, Zeiss) on the entire aorta cross section with the Axiovision Software Rel 4.7 (Zeiss). The percentage of positive area was defined as the ratio between IL6 positive area to the total area of the aortas.
The quantification of IL6 signal was carried after acquiring the images with an Axioskop II microscope (Zeiss) using a digital camera (AxioCam Color, Zeiss) on the entire aorta cross section with the Axiovision Software Rel 4.7 (Zeiss). The percentage of positive area was defined as the ratio between IL6 positive area to the total area of the aortas.
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