The following monoclonal antibodies (mAbs) or isotype control were employed for cell marker detection: for endothelial cells, the anti-CD31 rabbit monoclonal antibody (mAb) EPR17259 (Abcam, 1:50 dilution); for epithelial marker detection, the anti-pan cytokeratin mAb AE1/AE3 (Abcam, 1:10 dilution). Cells were fixed with ice cold methanol (CD31) or 3% paraformaldehyde (cytokeratin). To permeabilize samples for cytokeratin detection, 0.2% Saponin (CalbioChem) in 1% BSA (Sigma)/PBS was employed. Samples were blocked with 2% goat serum plus 1% BSA in PBS and then incubated for 1 h with primary antibodies. Cells were then washed with 1% BSA in PBS and blocked with previously described serum. For detection, cells were probed with either Alexaflour (AF) 647 goat anti-rabbit IgG (H + L) (Invitrogen) or Alexaflour (AF) 647 goat anti-mouse IgG (H + L) (Invitrogen). After two washes, nuclei were stained with DAPI (1/1,000) (AAT Bioquest) for 5 min. All micrographs were taken with the EVOS FL Auto 2 (Invitrogen) at 10× using the 2.0 Imaging System. Images were enhanced with Adobe Photoshop CC 2015 software.
Epr17259
Manufactured by Abcam
Sourced in United Kingdom
EPR17259 is a monoclonal antibody that can be used for the detection of SARS-CoV-2 nucleocapsid protein. It is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Evos fl auto 2, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by AAT Bioquest (1 mentions)
Ae1 ae3, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Protein block solution, by Agilent Technologies (1 mentions)
Ab8295, by Abcam (1 mentions)
Cryostat, by Leica (1 mentions)
Epifluorescent microscope, by Olympus (1 mentions)
Ab184787, by Abcam (1 mentions)
Ab15580, by Abcam (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Saponin, by Merck Group (1 mentions)
Ab150083, by Abcam (1 mentions)
Isolectin griffonia simplicifolia af 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Ab28244, by Abcam (1 mentions)
Ab76104, by Abcam (1 mentions)
Ab182981, by Abcam (1 mentions)
Nanozoomer, by Hamamatsu Photonics (1 mentions)
4 protocols using epr17259
1
Immunofluorescence Staining of Endothelial and Epithelial Cells
2
Histological and Immunofluorescence Analysis of Murine Tissues
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Mice tissues were harvested and fixed using a neutral buffered 10% formalin solution (NBF, Sigma-Aldrich, St. Louis, MO, USA) overnight. Afterward, the tissues were dehydrated in a 30% sucrose solution at 4°C for at least one night. Subsequently, the tissues were cryopreserved in optimal cutting temperature (OCT) compound and cryosectioned (CryoStats, Leica). Pathological assessments were carried out using H&E and Masson’s trichrome staining in accordance with the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO, USA).
For immunofluorescence staining, cryosections of the tissues were first permeabilized and blocked using a protein block solution (Dako, Carpinteria, CA, USA) containing 0.1% saponin (Sigma-Aldrich, St. Louis, MO, USA). The primary antibodies were then added and incubated overnight at 4°C, including rabbit anti-Caspase 3 (1:100; ab184787, Abcam, Cambridge, UK), rabbit anti-Ki67 (1:100; ab15580, Abcam), anti-CD31 antibody (1:100; EPR17259, Abcam), and mouse Anti-Cardiac Troponin T antibody (1:100; ab8295, Abcam) to target proteins of interest. Fluorophore-conjugated secondary antibodies were then added for fluorescent imaging. All the tissue slides were mounted by ProLong gold antifade mountant with DAPI (Thermo Fisher, US) before being imaged using an epifluorescent microscope from Olympus.
For immunofluorescence staining, cryosections of the tissues were first permeabilized and blocked using a protein block solution (Dako, Carpinteria, CA, USA) containing 0.1% saponin (Sigma-Aldrich, St. Louis, MO, USA). The primary antibodies were then added and incubated overnight at 4°C, including rabbit anti-Caspase 3 (1:100; ab184787, Abcam, Cambridge, UK), rabbit anti-Ki67 (1:100; ab15580, Abcam), anti-CD31 antibody (1:100; EPR17259, Abcam), and mouse Anti-Cardiac Troponin T antibody (1:100; ab8295, Abcam) to target proteins of interest. Fluorophore-conjugated secondary antibodies were then added for fluorescent imaging. All the tissue slides were mounted by ProLong gold antifade mountant with DAPI (Thermo Fisher, US) before being imaged using an epifluorescent microscope from Olympus.
3
Histopathological Evaluation of Kidney Tissue
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Following hypothermic perfusion, the kidneys were cut in the coronal plane to expose the cortex and medulla for gross imaging, transferred to 10% Neutral Buffered Formalin and paraffin embedded within 48 h. Using a microtome, 5 µm sections were acquired for Hematoxylin & Eosin (H&E), Prussian blue and confocal microscopy. 40× magnification brightfield images were obtained (TissueScope LE, Huron Digital Pathology, St. Jacobs, Ontario). Sections were labelled with rabbit anti‐CD31 primary antibody (EPR17259, abcam, Cambridge, UK) and goat anti‐rabbit IgG H&L (AF‐647) secondary antibody (ab150083, abcam). The sections were also labelled with Isolectin Griffonia simplicifolia AF‐488 Conjugate (ThermoFisher, Waltham, MA) towards d ‐galactosyl residues of Galactose α‐1,3 Galactose (Gal α‐1,3 Gal). Nuclei were labelled with 4’, 6‐diamidina‐2‐phenylindole (DAPI).
4
Comparison of Tumor Angiogenesis Markers
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4 µm paraffin-embedded tumor serial sections were used to compare Prussian blue staining with CD31, PSMA, and FAP immunohistochemistry. Staining was performed using a rabbit-specific HRP/DAB detection kit (Abcam) following the manufacturer's instructions, with modifications. Briefly, after hydration through a series of ethanol, sections were treated with hydrogen peroxide blocking for 10 min. Antigen retrieval was performed using pH 6 10 mM citrate buffer at 98 °C for 30 min and then incubated with 10% normal goat serum for 1 h for protein blocking. The sections were incubated overnight at 4 °C with anti-CD31 antibody (1:500, Abcam ab182981, EPR17259), anti-FAP antibody (1:300, Abcam ab28244), or anti-PSMA antibody (1:500, Abcam ab76104, EP3253). After washing, sections were treated with biotinylated antibody and streptavidin peroxidase and developed with 3,3′-diaminobenzidine (DAB) (all provided in the kit). Images were processed using a Nanozoomer (Hamamatsu, Japan) and were visualized using ImageJ software (Bethesda, Maryland, USA).
The percentage area of positive staining was calculated by setting color thresholds using ImageJ. Entire images have been adjusted for brightness, contrast, and exposure, consistently between stains.
The percentage area of positive staining was calculated by setting color thresholds using ImageJ. Entire images have been adjusted for brightness, contrast, and exposure, consistently between stains.
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