Fixed monolayers were incubated for 18hrs at 4°C with Rhodopsin antibody alone (single-staining; 1:300 diluted) or in combination with trkANGFR or p75NTR antibodies (double staining; 1:100 diluted). To assess for staining specificity, parallel sections were incubated with purified non-specific rabbit IgG antibodies (isotype; Vector, Burlingame, CA, USA). After appropriate washing, the slides were incubated with Alexa Fluor-488 goat anti-rabbit and/or Alexa Fluor-594 anti-specie-specific IgG secondary antibodies (1:400) for 1hr at room temperature. Nuclear counterstaining was performed with DAPI (0.1mM; Molecular Probes, Eugene, OR, USA). Sections were mounted in home-made anti-fading medium, observed under a confocal microscope (CLSM; SP5 Leica Microsystems; Wetzlar, Germany) and acquired at X60/objective (1024x1024 pixels; 10Hz acquisition speed; 30 or 60 μm pine-hole and low/high laser exposure). No intensity-image adjustment was performed while for presentation purposes color–image changes were performed after assembling in panels (Adobe Photoshop ver.7; Adobe System, San Jose, CA, USA).
Sp5 microsystems
Manufactured by Leica
Sourced in Germany
The Leica SP5 is a confocal microscope system designed for advanced imaging applications. It provides high-resolution, multicolor fluorescence imaging capabilities. The SP5 features a modular design, allowing for customization to suit specific research needs.
Automatically generated - may contain errors
4 protocols using sp5 microsystems
1
Immunostaining of Membrane Receptors
2
Zebrafish Angiogenesis Imaging Protocol
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Zebrafish were grown and maintained according to protocols approved by the Yale University Animal Care. The Tg(kdrl:mCherry; flt4:citrine) was used (Bussmann and Schulte-Merker, 2011 (link)). Morpholinos (Nicoli et al., 2012 (link)) were injected at the indicated concentrations and morphants were observed in a confocal microscope (SP5 Leica Microsystems). Images captured using Leica application suite software. Chemical treatment with nifedipine 40 µM was performed as previously described, 4 hr prior imaging (Bussmann et al., 2011 (link)).
3
miRNA Gene Reporter Assay and Mutation Analysis
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The miRNA gene reporter assay was constructed as previously described26 (link). sgRNA (100 ng/μl), Cas9 (150 ng/μl) with or without the corresponding GFP-miR-24-MRE or GFP-Let-7-7-MREs (50 ng/μl) and mCherry (50 ng/μl) with Phenol Red were injected. 48 hpf embryos were imaged with a confocal microscope (SP5 Leica Microsystems) and captured using Leica application software suite. Mutant and wild type miRNA genome sequence were cloned as previously described31 (link). Northern blots were performed using 3 micrograms of total RNA extracted from mCherry positive injected embryos and developed as reported previously in26 (link).
4
Quantifying Collagen Content in Arterial Walls
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Type I and III collagen content was assessed in MA fixed in PFA 4% by immunofluorescence. Briefly, arteries were incubated with anti-type I or type III collagen antibodies (1:200, Abcam) for 60 min at RT. Segments were washed and incubated with Alexa Fluor 647® anti-rabbit IgG (1:200 dilution, 1h, RT, Molecular Probes). Finally, nuclei were stained with DAPI. After rinsing, MA segments were mounted as described above and visualized with a confocal microscope (SP5 Leica Microsystems) by using x40 objective zoom 2. A minimum of three regions, were randomly selected. To avoid biased selection, the regions were chosen in the DAPI wavelength, as described above. Once selected the images were acquired at identical conditions of brightness, contrast, and laser power at 633 nm excitation/-640-650 nm emission wavelength (secondary antibody-Alexa 647) to detect collagen, either type I or type III. Quantitative analysis of collagen content in vascular wall was performed with Metamorph images analysis software as follows. An extended focus image was reconstructed from the serial images. Thereafter, total and background fluorescence intensity values were measured in the reconstructed image.
Collagen content was then estimated by subtracting the background from the total intensity fluorescence values.
Collagen content was then estimated by subtracting the background from the total intensity fluorescence values.
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