The 2-µm slice specimens created from paraffin blocks underwent immunostaining with primary antibodies of CK7 (dilution time is 250:1, cat. #ab9021, Abcam, Tokyo, Japan), PDX1 (500:1, cat. #ab9021, Abcam) and CDX2 (250:1, cat. #ab76541, Abcam). Histofine MAX-PO (Nichirei, Tokyo, Japan), which is specialized for use on mouse tissue, was used to activate the antigens with heat treatment, and visualization was then performed using DAB (Nichirei).
Ab9021
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab9021 is a primary antibody product manufactured by Abcam. It is a specific antibody that can be used for the detection and analysis of target proteins in various biological samples and applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab52625, by Abcam (3 mentions)
Ab78196, by Abcam (3 mentions)
Nucblue fixed readyprobes reagent, by Thermo Fisher Scientific (3 mentions)
Triton x 100, by Merck Group (2 mentions)
Ab24590, by Abcam (2 mentions)
Ab85379, by Abcam (2 mentions)
Ab179804, by Abcam (2 mentions)
Ab92547, by Abcam (2 mentions)
Ab76126, by Abcam (2 mentions)
Ab668, by Abcam (2 mentions)
Ab15148, by Abcam (2 mentions)
Ab75769, by Abcam (2 mentions)
3 3 diaminobenzidine (dab), by Nichirei Biosciences (1 mentions)
Ab76541, by Abcam (1 mentions)
D9542, by Merck Group (1 mentions)
A12381, by Thermo Fisher Scientific (1 mentions)
Ab178566, by Abcam (1 mentions)
Ab191073, by Abcam (1 mentions)
A1 confocal microscope, by Nikon (1 mentions)
Sc 56, by Santa Cruz Biotechnology (1 mentions)
23 protocols using ab9021
1
Immunohistochemical Markers in Tissue
2
Immunofluorescence Characterization of Kidney Embryoid Bodies
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Control or met-IPSC kidney embryoid bodies cultured on 96-well culture dishes or 8-well culture chambers were washed with PBS, fixed with 4% paraformaldehyde in PBS for 120 min, permeabilized with 0.2% Triton X-100 (Sigma, St. Louis, USA) in PBS and blocked in 10% serum. Primary antibodies were diluted in PBS 10% serum at the following concentrations: Nephrin (ab85379, 1:100, Abcam, Cambridge, UK), CD31 (ab24590, 1:50, Abcam, Cambridge, UK), PODXL (ab178566, 1:100, Abcam, Cambridge, UK), LTL (FL-132, 1:50, Clinisciences, Nanterre, France), VHL (SC-5575, 1:30, Bio-Techne, Minneapolis, USA), Phalloidin (A12381, 1:100, Invitrogen, Carlsbad, USA), cytokeratin 7 (ab9021, 1:100, Abcam, Cambridge, UK), TFE3 (ab179804, 1:100, Abcam, Cambridge, UK), Cubilin (ab191073, 1:50, Abcam, Cambridge, UK), phospho-Met (3077S, 1:100, Ozyme, Saint-Cyr-l’École, France), KDM4C (LS-C114684-100, 1:50, LSBio, Seattle, USA), BHLHE40 (ab70723, 1:50, Abcam, Cambridge, UK) and then washed in PBS. The samples were incubated with fluorescent secondary antibodies in antibody dilution buffer, then washed in PBS. Nuclei were labeled with DAPI (D9542, Sigma–Aldrich, St. Louis, USA) mounting medium. Visualization and capture were realized with a Leica confocal microscope type SP5-AOBS (Leica, Wetzlar, Germany) and NIKON microscope (NIKON, Minato, Japan).
3
Immunohistochemical Analysis of Mouse Liver
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Paraffin-embedded mouse liver sections (3-µm thickness) were prepared by a routine procedure. After blocking with 10% donkey serum for 30 min, the slides were immunostained with primary antibodies against cytokeratin (CK)7 (ab9021), CK19 (ab52625) (Abcam), and proliferating cell nuclear antigen (PCNA) (sc-56; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). After washing, they were incubated with the secondary antibodies (Alexa Flour 488 and/or Alexa Fluor 594) for 30 min at 37°C. Sections were then counterstained with Hoechst 33342. Stained slides were imaged using a Nikon A1+ confocal microscope (Nikon).
4
Histological Analysis of Neobladder Tissues
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After physiology test, blood and urine sample collection, pigs were euthanized, and their neobladders were harvested for histological analysis. The harvested neobladder tissues were fixed in 10% formalin for 1 week, then embedded with paraffin and sliced into 5 μm sections. Haematoxylin and eosin (H&E) stains were performed according to standard procedures. Immunohistochemical (IHC) analysis was performed using the mouse monoclonal antibody against cytokeratin 7 (CK7) (1:100, ab9021, Abcam), Uroplakin III (UPK3) (1:20, ab78196, Abcam) and alpha smooth muscle actin (α-SMA) (1:200, A5228, Sigma-Aldrich). The staining was performed according to the manufacturer of the mouse and rabbit specific HRP/DAB detection IHC kit (ab64264, Abcam). Images were obtained using the Pannoramic Scanner system (3D HISTECH, Ltd., Budapest, Hungary).
5
Immunofluorescence Analysis of Pancreatic Markers
6
Immunoblotting of Inflammatory Markers
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Immunoblotting was performed as described elsewhere [11 (link)]. Antibodies and dilutions employed in this study include anti–TNF-α (1:2000; cat. # 3114560, Sony), anti–β-actin (1:2000; cat. # ab8226, Abcam), anti–α smooth muscle actin (α-SMA; 1:2000; cat. # ab7817, Abcam), anti–cytokeratin 7 (1:2000; cat. # ab9021, Abcam), and anti-CD68 (1:2000; cat. # ab201340, Abcam) antibodies. Quantitative densitometric analyses were performed on digitized images of immunoblots in the Quantity One software (Bio-Rad, USA).
7
Trophoblast and Mesenchymal Cell Identification
8
Antibody Validation for Cell Death Pathways
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The following antibodies were purchased from Santa Cruz Biotechnology, Dallas, TX, USA: cleaved caspase-8 (sc-1226), RIP3 (sc-47368), ACTB (sc-1616) and α-tubulin (TUBA; sc-31779). Cleaved caspase-3 (no. 9661) and RIP1 (no. 3493) were purchased from Cell Signaling Technology, Danvers, MA, USA. Antibodies specific to PLAP (ab118856), RIP3 (ab56164), total MLKL (ab194699), phospho-MLKL (ab187091 and its competing peptide ab206929), cytokeratin-7 (ab9021), and E-cadherin (ab1416) were purchased from Abcam, Cambridge, UK. GCM1 antibody (P100836_P050) was purchased from Aviva Systems Biology, Corp., San Diego, CA, USA. Secondary antibodies include horseradish peroxidase-conjugated donkey anti-goat, goat anti-rabbit and goat anti-mouse from Santa Cruz Biotechnology. For immunofluorescence, the following secondary antibodies were used: Alexa Fluor 594 donkey anti-rabbit and Alexa Fluor 488 donkey anti-rabbit from Life Technologies, Carlsbad, CA, USA.
9
Western Blot Analysis of Cellular Proteins
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Whole cell extracts were prepared with a cell lysis reagent (Sigma-Aldrich; Merck KGaA) according to the manual, and the protein was quantified by a BCA assay (Pierce; Thermo Fisher Scientific, Inc.). Protein extracts were separated using SDS/polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were hybridized with anti-β-catenin antibody (dilution 1:500, ab32572; Abcam), TGFR1 (dilution 1:2,000, ab31013; Abcam), CK7 (dilution 1:500, ab9021; Abcam), CK20 (dilution 1:1,000, ab76126; Abcam), UPIII (dilution 1:800, ab78196; Abcam) and anti-β-actin antibody (dilution 1:800, sc-47778; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), at 4°C overnight. After extensive washing three times for 10 min each in TBS-T (Sigma-Aldrich; Merck KGaA), secondary biotin-conjugated antibodies were added for 1 h at 22°C. Blots were again washed three times for 10 min each in TBS-T, and immunoreactive bands were developed by an avidin/biotin/peroxidase complex (Vectastain ABC Elite kit; Vector Laboratories lnc., Burlingame, CA, USA) and substrate (Vector NovaRED, Vectastain; Vector Laboratories lnc.).
10
Comprehensive Tissue Histology Protocols
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For H&E, Masson’s trichrome, and FAH stains, tissue samples were fixed in 10% neutral-buffered formalin (Azer Scientific, Morgantown, PA, USA), paraffin embedded, and sectioned (5 μm). Standard stains were performed with established protocols, whereas IHC for Ki-67 was performed using monoclonal mouse anti-Ki-67 M7240 (Dako, Santa Clara, CA, USA), and FAH was performed as previously described.47 (link) Images were quantified in Aperio ImageScope. For all other IHC stains, tissues were fixed in 4% paraformaldehyde for 4 h, infiltrated with 30% sucrose overnight, and embedded in optimal cutting temperature (OCT) compound for sectioning (5–7 μm). Sections were washed with PBS, blocked with 5% bovine serum albumin (BSA) for 30 min, incubated with primary antibody for 1 h and secondary antibody for 30 min, and counterstained with Hoechst. Images were captured with an Olympus IX71 microscope. The following primary antibodies were used: ER-TR7 and glutamine synthetase cytokeratin 7 (ab51824, ab64613, ab9021; Abcam, Cambridge, MA, USA); CD31 (MCA1746GA; Bio-Rad, Hercules, CA, USA); cytokeratin 18 (ab668, Abcam, Cambridge, MA, USA); and 10830-1-AP, Proteintech, Chicago, IL, USA); and cytokeratin 19 (ab84632; Abcam, Cambridge, MA, USA).
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