Recombinant Human ACE2 Protein (933-ZN) was purchased from R&D Systems (Minneapolis, MN, USA) and stored in a buffer according to the manufacturer’s instructions. Monoclonal mouse anti-human ACE2 Antibody (MAB933) was purchased from R&D Systems (Minneapolis, MN, USA). Human recombinant VE-cadherin extracellular domain (EC1-5) was produced in the laboratory. Peroxidase AffiniPure Goat Anti-Mouse IgG (H + L) (AB-2338447), Peroxidase AffiniPure Goat Anti-Rabbit IgG (H + L) (AB-2307391), and Cy3™ AffiniPure Goat Anti-Mouse IgG (H + L) (AB-2338680) were all purchased from Jackson Immunoresearch (Ely, Cambridgeshire, UK). Hoechst solution (33,258) was purchased from Sigma Aldrich. The mouse monoclonal anti-human VE-cadherin antibody (clone BV9) and the rabbit polyclonal anti-EC1 anti-human VE-cadherin antibody were produced in the laboratory.
Cy3 affinipure goat anti mouse igg h l
Manufactured by Jackson ImmunoResearch
Sourced in United Kingdom
Cy3™ AffiniPure Goat Anti-Mouse IgG (H + L) is a secondary antibody product designed for immunohistochemical and immunofluorescence applications. It is produced by immunizing goats with mouse immunoglobulins and is affinity-purified to ensure specificity.
Automatically generated - may contain errors
Lab products found in correlation
Peroxidase affinipure goat anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions)
Peroxidase affinipure goat anti mouse igg h l, by Jackson ImmunoResearch (1 mentions)
Hoechst solution 33 258, by Merck Group (1 mentions)
Hrp labeled goat anti mouse igg h l, by Beyotime (1 mentions)
Antifade mounting medium, by Beyotime (1 mentions)
Ab6671, by Abcam (1 mentions)
Ab191860, by Abcam (1 mentions)
P2297, by Merck Group (1 mentions)
Ab16502, by Abcam (1 mentions)
Hrp labeled goat anti rabbit igg h l, by Beyotime (1 mentions)
Confocal microscope, by Zeiss (1 mentions)
Mouse anti α actinin, by Merck Group (1 mentions)
Tunel fitc apoptosis detection kit, by Vazyme (1 mentions)
Ps473 akt, by Cell Signaling Technology (1 mentions)
Ps15 p53, by Cell Signaling Technology (1 mentions)
γh2ax, by Merck Group (1 mentions)
53bp1 nb100 305, by Novus Biologicals (1 mentions)
Phostop, by Roche (1 mentions)
Cy3 affinipure goat anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
5 protocols using cy3 affinipure goat anti mouse igg h l
1
Recombinant ACE2 and VE-cadherin Protein Analysis
2
Inflammatory Bowel Disease Pathways
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2,4,6-trinitrobenzenesulfonic acid (TNBS; P2297, Sigma), anti-IL-18 (ab191860, Abcam), anti-TNF-α (ab6671, Abcam), anti-NF-κB p65 (ab16502, Abcam), anti-microtubule-associated protein 1 Light chain 3 beta (LC3B; #3868, Cell Signaling Technology, CST), anti-sequestosome 1 (p62; #23214, CST), anti-Beclin-1 (#3738, CST), anti-phospho-mTOR (p-mTOR; #2971, CST), Cy3-AffiniPure goat anti-mouse IgG (H + L) (111-165-003, Jackson), HRP-labeled goat anti-rabbit IgG (H + L) (A0208, Beyotime), HRP-labeled goat anti-mouse IgG (H + L) (A0216, Beyotime), insulin (P3376, Beyotime), rapamycin (LC R-5000, LC Laboratories), anti-PI3K p85 (#4292, CST), anti-Akt (#9272, CST), anti-p-Akt (#9271, CST), anti-Vps34 (V9764, sigma), and Antifade mounting medium (Beyotime, P0126) were used in this study.
3
Quantifying Cardiomyocyte Apoptosis by TUNEL
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Terminal deoxynucleotidyl transferase-mediated dUTP in situ nick end labeling (TUNEL) staining was conducted to detect apoptotic nuclei by confocal microscopy in α-actinin-labeled cardiomyocytes as described before [21 (link)]. Briefly, neonatal rat cardiomyocytes (NRCMs) or frozen mouse heart sections were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.5% Triton X-100 in PBS, and blocked with 5% bovine serum album (BSA) before incubation with mouse anti-α-actinin (Sigma, A7811, 1:200 dilution). After incubated with Cy3-AffiniPure goat anti-mouse IgG (H+L) (Jackson), cells or tissue sections were stained with TUNEL FITC Apoptosis Detection Kit (Vazyme) according to the manufacturer’s instructions. Nuclei were counterstained with DAPI. Finally, 20–30 fields per sample were viewed under a confocal microscope (Carl Zeiss). The percentage of TUNEL-positive cardiomyocytes was calculated to determine apoptosis induced by OGDR or I/R injury.
4
Immunofluorescence Analysis of Prostate Tissue
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Prostate tissue samples were immediately fixed in ice-cold 4% formaldehyde supplemented with 1 tablet/10 ml of PhoSTOP (04 906 837 001; Roche). Prostate samples were embedded in paraffin, and 5-µm serial sections were cut. For histopathological analyses, paraffin sections were stained with H&E. For immunofluorescence staining, sections were processed as previously described (Ratnacaram et al., 2008 (link)), except that sections were incubated over night with primary antibodies diluted 1:200, unless indicated. Primary antibody used for immunofluorescence were directed against AKT pS473 (4060; Cell Signaling Technology), γH2AX (05-636; EMD Millipore; 1:600), p53 (CM5; Vector Labs), p53 pS15 (12571; Cell Signaling Technology), pHP1γ (2600; Cell Signaling Technology), RPA32 (GTX113004; Tebu-Bio), RPA32 pS4/S8 (A300-245A; Bethyl Laboratories), ATR (2790; Cell Signaling Technology), Ki67 (M7248; Dako), BrdU (600-401-C29; Rockland), ATM pS1966 (200-301-400; Rockland), Mdm2 (sc-965; Santa Cruz Biotechnology), Mdm2 pS166 (human)/pS163 (mouse; 3521; Cell Signaling Technology), PCNA (ab2426; AbCam), 53BP1 (NB100-305; Novus Biologicals). Secondary antibodies (CY3 AffiniPure goat anti–rabbit IgG [H+L], CY3 AffiniPure goat anti–mouse IgG [H+L], CY5 AffiniPure goat anti–mouse IgG [H+L], and CY5 AffiniPure donkey anti–rabbit IgG [H+L]) were from Jackson Immunoresearch.
5
Immunofluorescent Visualization of Tight Junctions
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The polarized Caco-2 monolayer was washed with HBSS thrice and fixed using 4% paraformaldehyde for 15 min. The transwell membranes were excised from their plastic fitting with a razor, and the cells were permeabilized with 0.1% saponin in phosphate-buffered saline (PBS) for 30 min. Occludin was detected using mouse anti-occludin antibody (Invitrogen, Carlsbad, CA) and Cy3 AffiniPure goat anti-mouse IgG (H+L) (Jackson ImmunoResearch, West Grove, PA). Actin was bound by Alexa Fluor 680 phalloidin (Thermo Fisher Scientific, Waltham, MA), and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Thermo Fisher). Cells were imaged by deconvolution microscopy in a Delta Vision microscope (General Electric, Boston, MA). 3D visualization was performed using IMARIS software (Bitplane, Zurich, Switzerland). Viral quantification and colocalization were assessed using on-board IMARIS utilities.
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