After 28 days of culture, cells within the scaffolds were fixed in formalin (10% phosphate buffer, Fisher Scientific) and characterized by IHC for the expression of OCN. Heat-mediated antigen retrieval was performed with sodium citrate buffer (pH 6.0) for 20 min at 95 °C. Endogenous peroxidase was blocked with 3% H2O2 in methanol for 10 min, and nonspecific binding was suppressed with 1% horse serum in PBS for 45 min. Following antigen retrieval and blocking, the samples were incubated with rabbit anti-OCN primary antibody (1:200) (Abcam ab93876, Cambridge, MA) or an isotype control antibody (Abcam) overnight at 4 °C, followed by 30 min of incubation with horse anti-rabbit biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA). Staining was developed by incubating the samples with horseradish peroxidase-conjugated streptavidin (Vector Laboratories) and treating the samples with the Vector® NovaRED™ peroxidase substrate (Vector Laboratories). Following IHC, the samples were counterstained with hematoxylin, dehydrated, mounted, and coverslipped, and images were captured with an Olympus CKX41 microscope (Center Valley, PA).
Isotype control antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
An isotype control antibody is a type of antibody used in flow cytometry and other immunoassays to help determine the specificity of a primary antibody. It is designed to have the same isotype as the primary antibody but does not bind to the target antigen. Isotype control antibodies are used to establish the level of non-specific binding and help distinguish specific from non-specific signals.
Automatically generated - may contain errors
Lab products found in correlation
Kaluza analysis software, by Beckman Coulter (2 mentions)
Accutase, by Innovative Cell Technologies (2 mentions)
Gallios flow cytometer, by Beckman Coulter (2 mentions)
Perfix expose kit, by Beckman Coulter (2 mentions)
Lsrfortessa, by BD (2 mentions)
Formalin, by Thermo Fisher Scientific (1 mentions)
Rabbit anti ocn primary antibody, by Abcam (1 mentions)
Horse anti rabbit biotinylated secondary antibodies, by Vector Laboratories (1 mentions)
Horseradish peroxidase conjugated streptavidin, by Vector Laboratories (1 mentions)
Vector novared peroxidase substrate, by Vector Laboratories (1 mentions)
Ckx41 microscope, by Olympus (1 mentions)
Cytofix cytoperm buffer, by Thermo Fisher Scientific (1 mentions)
Antibodies against foxp3, by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Facs canto 2 instrument, by BD (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Golgiplug, by BD (1 mentions)
4 6 diamidino 2 phenylindole dapi containing mounting medium, by Vector Laboratories (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Ix81 microscope, by Olympus (1 mentions)
12 protocols using isotype control antibody
1
Immunohistochemical Analysis of Osteocalcin Expression
2
Quantifying Cell Surface Markers
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Cells were collected using accutase (Innovative Cell Technologies, San Diego, CA) and stained with mouse monoclonal TDM29 anti-CD29 (diluted 1:50; Sigma-Aldrich), rat monoclonal IM7 anti-CD44 (diluted 1:20; ThermoFisher Scientific), or rat monoclonal GoH3 anti-CD49f (diluted 1:10; BD Biosciences, San Jose, CA) antibodies diluted in PBS with 1% BSA, for 1 h. Staining with an isotype control antibody (Abcam), diluted 1:10 in PBS with 1% BSA, or no antibody stain were included as controls. Cells were washed three times in PBS and incubated with Alexa 488-conjugated secondary goat anti-rabbit and goat anti-mouse antibodies diluted 1:500 in PBS with 1% BSA, for 30 min. After washing, 50,000 cells were analyzed on a Gallios flow cytometer controlled by Kaluza for Gallios software (Beckman Coulter, USA). Data analysis was conducted using Kaluza Analysis software (Beckman Coulter).
3
Intracellular Cytokine and PRMT5 Staining
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For intracellular cytokine staining, cells were stimulated with 50 ng/ml PMA and 500 ng/ml ionomycin (Sigma-Aldrich) in the presence of GolgiPlug (BD Biosciences) for 5 h. Then, the cells were fixed and permeabilized with Cytofix/Cytoperm buffer, and intracellular cytokines were stained with antibodies against Foxp3, IFN-γ, and IL-2 (eBioscience). For PRMT5 staining, the cells were stained with an antibody or isotype control antibody (Abcam, 1/100 dilution) for 30 min at room temperature according to the manufacturer’s instructions. Flow cytometric analysis was performed with a FACS Canto II instrument (BD Bioscience) and FlowJo software (TreeStar).
4
Muscle Tissue Histological Analysis
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Muscle tissues were fixed in methanol, paraffin embedded, sectioned, and processed for routine haematoxylin and eosin, Trichrome, and Alizarin Red staining. Immunohistochemistry (IHC) was performed according to protocols described for the Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, California, USA). Primary antibodies against phosphorylated Smad2, fibrin(ogen), as well as isotype control antibody were purchased from Abcam. Antibodies were used in a 1:100 to 200 dilution range at a concentration of 5 μg/ml. For immunofluorescence staining, primary antibody against platelet-derived growth factor receptor α (PDGFRα) (AF1062; R&D Systems, Minneapolis, Minnesota, USA) was used to detect the presence of MDSCs. The slides were mounted with 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting medium (Vector Laboratories), and were viewed using an inverted IX81 microscope (Olympus, Tokyo, Japan) equipped with a Retiga EXi cooled CCD camera (Teledyne Qimaging, Surrey, Canada) and MetaMorph software (Molecular Devices, San Jose, California, USA).
5
Quantifying ATF4 expression in cells
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The cells were fixed and permeabilized using PerFix EXPOSE kit from Beckman Coulter (#PN B26976), according to the manufacturer's protocol. The cells were stained for 30 min at room temperature with the primary antibody against ATF4 conjugated with fluorochrome - Allophycocyanin using Zenon labelling kit (Life Technologies) appropriate for the antibody isotype. Isotype control antibody (Abcam) was labelled and used as a background staining control. Flow cytometry analysis was performed using LSR Fortessa (BD).
6
Quantification of PAD2 and PAD4 Expression
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Cells were collected using accutase (Innovative Cell Technologies, San Diego, CA), fixed for 15 min in 4% paraformaldehyde (PFA), permeabilized and blocked in 0.1% Triton-X with 1% normal goat serum in PBS with 1% bovine serum albumin BSA for 1 h, and stained with PAD2 or PAD4 antibodies, diluted 1:50 in PBS with 1% BSA, for 1 h. Staining with an isotype control antibody (Abcam), diluted 1:50 in PBS with 1% BSA, or no antibody stain were included as controls. Cells were washed three times in PBS and incubated with Alexa 488-conjugated secondary goat anti-rabbit antibodies diluted 1:500 in PBS with 1% BSA, for 30 min. After washing, 10,000 cells were analyzed on a Gallios flow cytometer controlled by Kaluza for Gallios software (Beckman Coulter, Indianapolis, IN). Data analysis was conducted using Kaluza Analysis software (Beckman Coulter, Indianapolis, IN).
7
Multimodal Characterization of Liver Cells
8
Kidney Histopathology and Immunohistochemistry
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Mice were anesthetized using a zoletil-xylazine cocktail during perfusion with phosphate-buffered saline (PBS). Kidney was vertically dissected and fixed with 10% formalin. Kidney sections (3 μm in thickness) were stained with Periodic acid-Schiff (PAS) stain (Sigma). Kidney pathology was evaluated using a lupus nephritis classification system as described in a previous study (16 (link)). Immunohistochemistry was performed using a Vectastain ABC kit (Vector). Tissue sections were incubated with anti-nephrin (Progen), anti-synaptopodin (Abcam), anti-podocin (Abcam), or isotype control antibodies (Abcam) at 4°C overnight. Staining was developed using 3,3′-diaminobenzidine chromogen (Dako). Sections were counterstained with hematoxylin QS (Vector).
9
PD-L1 Surface Expression Analysis
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Twenty-four hours after treatment initiation, the media were removed, and the cells were washed with ice-cold phosphate-buffered saline (PBS). The cells were trypsinized, collected, and washed with PBS to remove residual trypsin and subsequently incubated with PE Anti-PD-L1 antibody (ab270652, Abcam) or isotype control antibodies (Abcam, Cambridge, UK) for 30 min at 4°C. Next, they were washed with PBS before being fixed in 1 % paraformaldehyde. PD-L1 surface expression was measured using a BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed with FlowJo v10.6.2 (FlowJo LLC, Ashlang, OR USA), and all experiments were conducted in triplicates.
10
Characterizing BRCA and PALB2 Protein Levels
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