Sections from experiments 1, and 4 were analyzed at high power to study the neuronal connectivity of CART-IR neurons. In an additional quadruple-labeling study, the primary antibody cocktail from experiment 1 was supplemented with a polyclonal guinea pig GnRH antiserum (#1018; 1∶3000) [25] (link). This was followed by the cocktail of secondary antibody-fluorochrome conjugates: anti-guinea pig-AMCA, 1∶50; anti-mouse-FITC, 1∶250; anti-sheep-Cy3, 1∶1000; anti-rabbit-Cy5, 1∶500 (Jackson ImmunoResearch Laboratories) for 5 h. The visualization of KP, NKB and CART was carried out as in experiments 1 and 2. The AMCA signal for GnRH was overlaid to this three-color image in a separate Adobe Photoshop layer (screen mode), after being converted to a brown pseudocolor.
Anti sheep cy3
Manufactured by Jackson ImmunoResearch
Anti-sheep-Cy3 is a secondary antibody conjugated with the fluorescent dye Cyanine 3 (Cy3). It is designed to detect and visualize sheep primary antibodies in various immunoassays and imaging techniques.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit cy5, by Jackson ImmunoResearch (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Anti rabbit cy2, by Jackson ImmunoResearch (1 mentions)
Bp151 100, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Anti rabbit alexa 568, by Thermo Fisher Scientific (1 mentions)
Anti mouse alexa 488, by Thermo Fisher Scientific (1 mentions)
Ahp500g, by Bio-Rad (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
4 protocols using anti sheep cy3
1
Multiplex Visualization of Neuronal Connectivity
2
Immunostaining of Endothelial Markers
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Human tissue slides were removed from a −80 °C freezer, postfixed in ice-cold methanol for 10 min, and briefly washed in 0.1 M PBS. Sections were incubated for 2 h in 5% normal donkey serum (NDS) and 0.1% Triton X-100 (Fisher Bioreagents, BP151-100) in 0.1 M PBS for 2 h, before being incubated overnight at 4 °C with primary antibodies (rabbit anti-Cldn5, 1:250, Thermo Fisher Scientific, 34-1600 and sheep anti-hCD31, 1:250, R&D Biosystems, AF806). Slices were washed three times in 0.1 M PBS for 5 min and incubated with anti-rabbit Cy2 and anti-sheep Cy3 secondary antibodies for 2 h at room temperature (1:400, Jackson Immunoresearch, 711-225-152, 713-165-147, respectively). Sections were again washed three times in 0.1 M PBS and counterstained with DAPI to visualize nuclei. Slices were mounted and coverslipped with ProLong Diamond Antifade Mountant (Invitrogen, P36961). Images were acquired from 6-μm flattened z-stacks on a LSM-700 confocal microscope (Carl Zeiss) using a 20× lens with a resolution of 512 × 512 and a zoom of 1.0.
3
Identifying CART-IR and SP-IR Subpopulations
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To determine whether the CART-IR and the SP-IR subpopulations of KP and NKB neurons are identical or different, the primary antibody cocktail used in experiments 1 and 2 was supplemented with a rat monoclonal SP antibody (Serotec #8450-0505; Bio-Rad Laboratories, Inc., Hercules, CA; 1∶3,000) [33] (link). Following a 48-h-incubation in this mixture at 4°C, a cocktail of four different secondary antibody-fluorochrome conjugates were applied to the sections for 5 h at room temperature: anti-rat-AMCA, 1∶50; anti-mouse-FITC, 1∶250; anti-sheep-Cy3, 1∶1000; anti-rabbit-Cy5, 1∶500 (ea. from Jackson ImmunoResearch Laboratories). The visualization of the first three neuropeptides (KP, NKB and CART) was carried out as in experiments 1 and 2, whereas the SP signal was illustrated separately in a parallel black-and-white image. The presence/absence of quadruple-labeled neuronal elements was determined by switching between the superimposed Photoshop layers.
4
Immunofluorescence Staining of Golgi and Membrane Proteins
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Cells were grown on glass coverslips for one day, then fixed with 3% PFA for 10 min, quenched with 50 mM NH4Cl in PBS for 5 min, permeabilized with 0.2% TritonX-100 in PBS for 10 min, blocked with 1% BSA in PBS for 1 h, and incubated with primary antibodies diluted in 1% BSA in PBS for 1 h: Anti-AP1g1 (1:1000, self-made from hybridoma cells), anti-bCOP (1:500, CM1, hybridoma supernatant, gift from Dr. Felix Wieland, Heidelberg University), anti-GGA2 (1:500, BD Bioscience 612613), anti-GM130 (1:1000, Cell Signaling 12480S), and anti-TGN46 (1:1000, BioRad AHP500G). Samples were washed and incubated with fluorescent secondary antibodies diluted in 1% BSA in PBS for 1 h (1:400; anti-mouse-Alexa488, Invitrogen A21202; anti-rabbit-Alexa568, Invitrogen A10042; anti-sheep-Cy3, Jackson ImmunoResearch 713-165-147). Coverslips were mounted in FluoromountG (SouthernBiotech) supplemented with 0.5 ng/ml DAPI (Sigma-Aldrich) and stored in the dark at 4°C. For localization of GFP-KDEL, cells were grown on coverslips for one day, transfected with the pcDNA3-ss-GFP-KDEL, and fixed and stained a day later.
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