The peptide that contains 15 amino acids (AILFWQYFVGKRTVP) of the second extracellular loop of the M3 muscarinic acetylcholine receptor (M3R) protein (synthesized by Biomatic Corporation) was dissolved in PBS. After further diluted in 1×BioLegend ELISA coating buffer, this M3R peptide solution (2μg/ml) was used to coat Nunc™ MaxiSorp™ flat-bottom 96 well plates (BioLegend). Non-specific binding sites on the plates were blocked with ELISA Assay Diluent Buffer (BioLegend), and 1:6-diluted serum samples were added to the plates and incubated at 4°C overnight. The serum samples bound to the plates were then incubated with 1:300 diluted biotinylated goat anti-mouse IgG antibody (Vector Laboratories) for 1 h, followed by avidin-HRP solution for 30 min, and finally with the TMB substrate. The absorbance was measured at 450 nm using a BioTek microplate reader.
Biotinylated goat anti mouse igg antibody
Manufactured by Vector Laboratories
Sourced in United States
The Biotinylated goat anti-mouse IgG antibody is a secondary antibody used in various immunological techniques. It is produced by immunizing goats with mouse immunoglobulin G (IgG) and then conjugating the resulting antibodies with biotin. This antibody can be used to detect and localize mouse IgG in samples.
Automatically generated - may contain errors
Lab products found in correlation
Microplate reader, by Agilent Technologies (3 mentions)
Elisa assay diluent buffer, by BioLegend (3 mentions)
Vectastain abc kit, by Vector Laboratories (3 mentions)
Streptavidin hrp, by Agilent Technologies (2 mentions)
Dab chromogen, by Agilent Technologies (2 mentions)
Elisa coating buffer, by BioLegend (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Tetramethylbenzidine tmb substrate, by Thermo Fisher Scientific (1 mentions)
Streptavidin alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
35 mm confocal dish, by SPL Life Sciences (1 mentions)
Hoechst 33342 dye, by Thermo Fisher Scientific (1 mentions)
Methyl green, by Vector Laboratories (1 mentions)
Hematoxylin, by Vector Laboratories (1 mentions)
Abc elite, by Vector Laboratories (1 mentions)
Vector vip substrate, by Vector Laboratories (1 mentions)
Anti aromatase, by Abcam (1 mentions)
Ab97959, by Abcam (1 mentions)
Biotinylated goat anti rabbit igg antibody, by Vector Laboratories (1 mentions)
Mab5428, by Merck Group (1 mentions)
T2762, by Thermo Fisher Scientific (1 mentions)
15 protocols using biotinylated goat anti mouse igg antibody
1
M3 Muscarinic Receptor Peptide ELISA
2
Quantification of Anti-Laronidase IgG Antibodies
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Serum IgG anti-laronidase antibody levels were determined using a sandwich ELISA. Briefly, EIA plates were coated with 5 μg/mL laronidase diluted in coating buffer (0.1 M NaHCO3 [pH 8.5]) overnight at 4°C. Plates were washed with wash buffer (PBS and 0.1% Tween) and blocked with blocking buffer (1% BSA, 0.02 M Tris/HCl, and 0.25 M NaCl) for 1 h at room temperature. The mouse anti-human IDUA antibody (antibodies-online.com , Aachen, Germany), standards, and the serum samples were diluted in dilution buffer (0.05% Tween and 0.01% BSA in PBS). Plates were washed; standards and serum samples were applied to the plate in duplicate for 1 h at room temperature. Plates were washed and incubated with biotinylated goat anti-mouse IgG antibody (Vector Laboratories, Peterborough, UK) at 5 μg/mL for 1 h at room temperature. The plates were washed and then incubated with Vectastain ABC kit (Vector Laboratories, Peterborough, UK) for 30 min at room temperature, before incubating in 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific, Waltham, MA, USA) for exactly 3 min. 2.5 M H2SO4 was added to the plate to stop the reaction. Light absorbance was read at 450 nm to determine the maximum absorbance and at 570 nm to correct for measurement errors. The anti-laronidase IgG antibody concentrations of the serum samples were determined using the standard curve.
3
Immunofluorescence Assay for PEDV Infection
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Vero cells were maintained in α-MEM media supplemented with 5% heat-inactivated FBS and 1% antibiotic-antimycotic agent, and cultivated on coverslips placed in 35 mm confocal dish (SPL, Korea) for the immunofluorescence assay. After cells reached confluence, PEDV SM98 strain was inoculated onto the cell monolayer at multiplicity of infection (MOI) of 1 and incubated for 1 h at 37 °C. Subsequently, serum-free α-MEM media containing trypsin (2.5 μg/ml) was added and the culture was incubated for 3 days at 37 °C. Then, cells were fixed with 4% paraformaldehyde for 10 min at room temperature. Cells were subsequently blocked with 1% bovine serum albumin in PBS for 1 h at room temperature and then incubated with mouse anti-S25-229 immune sera for 1 h at room temperature. Then, cells were incubated with biotinylated goat anti-mouse IgG antibody at a dilution of 1: 200 (Vector, USA) followed by streptavidin-Alexa Fluor 488 (Invitrogen, USA) at a dilution of 1: 500. Finally, cells were counterstained with Hoechst 33342 dye (Invitrogen, USA) for 1 min, and cell staining was visualized using a confocal laser scanning microscopy LSM710 (Carl Zeiss, USA).
4
IgG Antibody Quantification Against AAV Capsids
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Total IgG antibody responses against AAV capsid proteins were measured with an enzyme-linked immunosorbent assay (ELISA) assay, using several brain homogenate dilutions, biotinylated goat anti-mouse IgG antibody (Vector) and the Vectastain ABC kit (Vector) (Supplementary material ).
5
Immunohistochemical Analysis of Aromatase Expression
6
Antibodies Used for Immunodetection in Newt Tissues
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The primary and secondary antibodies used for immunoblotting and immunohistochemistry were as follows: for Pax6, mouse monoclonal anti-Pax6 antibody (1:100; AD2.38, sc-32766; Santa Cruz Biotechnology, TX 75220, U.S.A) and biotinylated goat anti-mouse IgG antibody (1:500; Vector Laboratories, CA 94010, U.S.A); for Sox2, rabbit anti-Sox2 antibody (1:660; ab97959; Abcam, Cambridge, U.K.) and biotinylated goat anti-rabbit IgG antibody (1:500; Vector Laboratories); for RPE65, mouse monoclonal anti-RPE65 antibody (1:500; MAB5428; Millipore, Darmstadt, Germany) and tetramethylrhodamine-conjugated goat anti-mouse IgG antibody (1:200; T2762; Life Technologies, MD 20850, U.S.A); and for PCNA, human autoantibody to PCNA (1:500; gift from Dr. T. Saito)5 (link) and Alexa Fluor 488-conjugated goat anti-human IgG (1:1,000; A-11013; Life Technologies). For the negative control, mouse or rabbit antibodies (IgG) with no specific reactivity to newt tissues (e.g., anti-dsRed antibody; 632543 or 632496; Clontech, CA 94043, U.S.A) were applied, instead of the primary antibody, at the same concentration of IgG.
7
Quantifying Anti-M3R Antibody Levels
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The M3R peptide (AILFWQYFVGKRTVP) was synthesized by Biomatic Corporation and kindly provided by Dr. Toshihisa Kawai (Nova Southeastern University). The M3R peptide solution (1 µg/mL) was used to coat Nunc™ MaxiSorp™ flat-bottom 96-well plated (BioLegend) and incubated at 4 °C overnight. Non-specific binding sites on the plates were blocked with ELISA Assay Diluent buffer (BioLegend). Sera with serial dilution (1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560, 1:5120) were added to the plate and incubated at 4 °C overnight. The plates were incubated with biotinylated goat anti-mouse IgG antibody (Vector Laboratories, Newark, CA, USA), Avidin-HRP solution and TMB substrate followed by reading as previously reported [28 (link)]. The absorbance was measured at 450 nm and 570 nm with a microplate reader (BioTek, Santa Clara, CA, USA), and the absorbance at 450 nm subtracted by that at 570 nm was considered as an indicator for anti-M3R antibody quantity.
8
Immunohistochemical Analysis of Cell-Seeded Matrices
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Cell-seeded matrix samples were analyzed using immunohistochemistry. The samples were fixed in 10% buffered formalin and embedded in paraffin. After deparaffinization and high temperature citrate-based antigen retrieval, the slides were blocked with BLOXALL solution (Vector Laboratory Inc., Burlingame, CA, USA) and 5% goat serum. The slides were then washed in PBS and incubated in anti-cytokeratin antibodies (AE1/AE3, GA053, Dako, CA, USA) and incubated with the secondary antibody, i.e., Biotinylated Goat Anti-Mouse IgG Antibody (Vector Laboratory Inc., Burlingame, CA, USA) diluted at 1:400 for 30 min at room temperature. Finally, after incubation with the ABC reagent, i.e., Vectastain ABC Kit (Vector Laboratory Inc., Burlingame, CA, USA), slides were treated with DAB substrate, i.e., ImmPACT DAB Substrate Kit (Vector Laboratory Inc. Burlingame, CA, USA) and counterstained with hematoxylin.
9
CD44 Antibody Labeling and Cell Sorting
10
Double Immunoelectron Microscopy of GFAP and Reelin
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For double immunoelectron microscopy, sections were cryoprotected and freeze-thawed. After blocking in 20% NGS in 50 mM TBS, sections were incubated with primary antibodies for GFAP (rabbit, polyclonal, DAKO) and Reelin (mouse-monoclonal, clone G10, Millipore, Billerica, MA, USA) in 50 mM TBS containing 3% NGS (Vector Laboratories, Burlingame, CA) for 24h at 4°C. After washes in TBS, the sections were incubated with biotinylated goat anti-mouse IgG antibody (1:100; Vector Laboratories) and goat anti-rabbit (Fab fragment, 1:100) coupled to 1.4 nm gold (Nanoprobes, Stony Brook, NY). Subsequently, sections were processed for silver enhancement of the gold particles with an HQ Silver kit (Nanoprobes) and incubated with avidin-biotin peroxidase complex (ABC kit; Vector Laboratories) that was visualized with 3,3′-diaminobenzidine tetrahydrochloride (0.05%) as a chromogen and 0.01% H2O2 as substrate. Sections were then treated with 1% osmium tetroxide and uranyl acetate, dehydrated and flat-embedded in epoxy resin (Durcupan ACM Fluka; Sigma-Aldrich). Ultrathin sections were cut at 60–70 nm on an ultramicrotome (Reichert Ultracut E; Leica), and viewed on a Philips CM100 electron microscope. Images were taken with a CCD camera (Orius SC600; GATAN) and analyzed using GATAN imaging software.
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