Eyeballs were fixed in 1% PFA (paraformaldehyde) for 1 h to fix choroidal vasculature. We washed away the PFA with PBS and produced the RPE/choroidal/scleral whole mounts by separating the conjunctivae, corneas, lenses, and retinas. The mounts were incubated for 2 h at 37°C, and the RPE was washed away with PBS. The mounts were fixed in 1% PFA for 1.5 h again. After washing the PFA, the mounts were blocked using 0.5% Triton X-100/5% BSA overnight and incubated with the primary antibodies of ET-1 (1∶100; ab2728, Abcam, MA, USA) and CD31 (1∶100; MAB1398Z, Millipore, MA, USA) for 2 days at 4°C. The secondary antibodies of anti-mouse IgG antibody and anti-hamster IgG antibody (1∶800; Alexa Fluor® 488 Conjugate and Alexa Fluor® 647 Conjugate, CST, USA) were added for 1 h. The mounts were photographed using a Zeiss confocal microscope (LSM710, Carl Zeiss).
Ab2728
Manufactured by Abcam
Sourced in United States
Ab2728 is a rabbit polyclonal antibody targeting Abcam's proprietary antigen. This antibody is designed for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab28052, by Abcam (2 mentions)
Lsm 710, by Zeiss (1 mentions)
Mab1398z, by Merck Group (1 mentions)
Ab55667, by Abcam (1 mentions)
Sc 6465, by Santa Cruz Biotechnology (1 mentions)
Sc 15363, by Santa Cruz Biotechnology (1 mentions)
Ab2864, by Abcam (1 mentions)
Ab2861, by Abcam (1 mentions)
Ab3516, by Abcam (1 mentions)
Ab27642, by Abcam (1 mentions)
Ab16478, by Abcam (1 mentions)
Ab2865, by Abcam (1 mentions)
Ab9485, by Abcam (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
5 protocols using ab2728
1
Visualizing Choroidal Vasculature and ET-1
2
Isolation and Analysis of Hepatoma Cell-Derived Extracellular Vesicles
3
Cardiac Protein Expression Analysis
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Crude tissue homogenate from cardiac ventricles was separated by electrophoresis and transferred onto PVDF membranes. Membranes were incubated with primary antibodies: anti‐PAD2 (1:1000, Abcam, ab16478), anti‐RyR2 (1:5000, Abcam, ab2728), anti‐phosphorylated‐S2808‐RyR (1:5000, donated by Andrew R Marks, Columbia University), anti‐dihydropyridine receptor (1:1000, Abcam, ab2864), anti‐calsequestrin 2 (1:1000, Abcam, ab3516), anti‐SR Ca2+‐ATPase 2a (SERCA 2a; 1:1000, Abcam ab2861), anti‐phospholamban (1:1000, AbCam, ab2865), α‐actin (1:1000, Abcam ab28052), anti‐citrulline antibody (1:1000, Millipore, 07–377), Malondialdehyde (MDA) antibody (1: 1000, Abcam, ab27642), GAPDH (1:1000, Abcam, ab9485), and infrared‐labeled secondary antibodies (1:5000, Licor IRDye 680 and IRDye 800). Immunoreactive bands were analyzed using the Odyssey Infrared Imaging System. Band densities were quantified with Image J and normalized to GAPDH. All the experiments were conducted blinded regarding the treatment of the mice.
4
Immunoprecipitation of Cardiac Proteins
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Dynabeads Protein G (Invitrogen) were incubated with anti‐α‐actin (Abcam, ab28052) or RyR2 antibody (Abcam, ab2728) for 40 min at RT to allow the cross‐binding between beads and antibody, then the beads were washed three times with phosphate buffer solution (PBS). Cardiac ventricle lysate (500 μg) was incubated at 4°C overnight with the beads conjugated to the anti‐α‐actin antibody. The immunocomplex was washed once with lysis buffer and then three times with PBS. Finally, the immunoprecipitate was collected in 50 μl of Laemmli buffer 2× (Bio‐Rad #161–0737, with β‐Mercaptoethanol 10% and DTT 0.05 M). Proteins were separated by electrophoresis and immunoblots were performed as described above and quantified relative to the RyR2 or α‐actin expression.
5
Quantifying Endothelin-1 in RPE/Choroid
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After enucleating the eyeballs and cutting off conjunctivae, the RPE/choroidal/scleral complexes were isolated by removing the corneas, lenses, and retinas. The proteins in these complexes were homogenized in RIPA lysis buffer (Beyotime Institute of Biotechnology, China). The protein concentration was determined, and 20 μg of proteins from every group was used for the following procedures, as previously reported [16 (link)]. The primary antibody of mouse anti-endothelin 1 antibody (1∶1000; ab2728, Abcam, MA, USA) and the secondary antibodies of anti-mouse IgG antibody (1∶1000; 7076#, CST, USA) were used, and the gray intensity was estimated using a ChemiDoc™ MP Imaging System (BioRad, USA).
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