Transduced SOX2-deleted NSCs were dissociated to single cells and seeded on Matrigel™-coated glass coverslips at a density of 80,000 cell/coverslip. After 4 h, cells were fixed for 20 min with 4% PFA in phosphate-buffered saline (PBS; pH 7.4) and rinsed three times with PBS. Coverslips were then incubated for 90 min in PBS containing 10% normal goat serum, 0.2% Triton-X100 at room temperature. Coverslips were then incubated with the primary anti-SOX2 antibody (mouse monoclonal IgG2a, 1:100, R&D Systems), overnight at 4 °C. After thorough washing with PBS, cells were incubated for 45 min at room temperature with secondary goat anti-mouse IgG2a cross-adsorbed secondary antibody, Alexa Fluor 594, (Thermo Fisher Scientific, Catalog# A-21135, 1:1000). Coverslips were rinsed three times in PBS and mounted on glass slides with Fluoromount (Sigma) with DAPI (4′,6-diamidino-2-phenylindole).
Anti mouse igg2a
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
Anti-mouse IgG2a is a laboratory reagent used to detect and measure the presence of the IgG2a subclass of mouse immunoglobulin in experimental samples. It functions as a specific binding agent for the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Elisa plate reader, by Tecan (2 mentions)
Anti mouse igg2b, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Fluoromount, by Merck Group (1 mentions)
Hrp conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
3 3 5 5 tetramethylbenzidine tmb substrate, by Merck Group (1 mentions)
Vectra 3, by PerkinElmer (1 mentions)
Grp 78 3177, by Cell Signaling Technology (1 mentions)
Kdel 10c3, by Enzo Life Sciences (1 mentions)
Perk 5683, by Cell Signaling Technology (1 mentions)
Reovirus μ1c 10f6, by Developmental Studies Hybridoma Bank (1 mentions)
Reovirus σ3 4f2, by Developmental Studies Hybridoma Bank (1 mentions)
Hrp conjugated anti mouse igg, by Fortis Life Sciences (1 mentions)
3 3 5 5 tetramethylbenzidine tmb substrate, by Thermo Fisher Scientific (1 mentions)
Cryostar nx70, by Thermo Fisher Scientific (1 mentions)
Fsc 22 clear frozen section compound, by Leica (1 mentions)
Ab5694, by R&D Systems (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Mab1195, by R&D Systems (1 mentions)
Fluoromount, by Diagnostic BioSystems (1 mentions)
6 protocols using anti mouse igg2a
1
Immunofluorescent Analysis of SOX2 Expression
2
MERS-CoV Antibody Detection by ELISA
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Enzyme-linked immunoassay (ELISA) was carried out to detect specific antibodies in mouse sera as described below (Tai et al., 2016a (link)). Specifically, ELISA plates were coated with MERS-CoV S1-His-protein (1 μg/ml) overnight at 4 °C, and blocked with 2% fat-free milk in PBST for 2 h at 37 °C. The plates were washed with PBST, and incubated sequentially with mouse sera at serial dilutions and horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:5000) (Invitrogen), anti-mouse IgG1 (1:5000) (Thermo Fisher Scientific), anti-mouse IgG2a (1:5000) (Thermo Fisher Scientific), and anti-mouse IgM (1:4000) (Invitrogen) antibodies, respectively, for 1 h at 37 °C. This step was followed by sequential incubation of the plates with TMB (3,3′,5,5′-tetramethylbenzidine) substrate (Sigma) and termination reagent (H2SO4, 1 N) to stop the reaction. Absorbance (at 450 nm) was measured by an ELISA plate reader (Tecan).
3
Multimodal Imaging of Tumor Spheroids
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Spheroids were rinsed in PBS before fixation in 10% neutral-buffered formalin (NBF). Spheroids were embedded in HistoGel before paraffin embedding and sectioning. In vivo tumors were fixed in NBF before paraffin embedding and sectioning. Heat-induced antigen retrieval was performed using pH 6 sodium citrate buffer. Tissue was blocked using 5% BSA before incubation with the following antibodies: PERK #5683, GRP78 #3177, and PDI #3501 were from Cell Signaling Technologies (MA, USA); KDEL 10C3 was from Enzo (Exeter, UK); and reovirus μ1C 10F6 and reovirus σ3 4F2 were from the Developmental Studies Hybridoma Bank (IA, USA). Alexa Fluor 488, 546, and 647 conjugates of anti-rabbit IgG H+L, anti-mouse IgG H+L, anti-mouse IgG2a, and anti-mouse IgG2b were from Thermo Fisher (UK). Spheroid sections were imaged on a Zeiss 710 confocal (Jena, Germany) with quantification using Cell Profiler v.3. Exposure settings were based on the minimum possible, which still allowed robust spheroid identification for segmentation. Controls were compared during exposure setup to ensure similar intensities across experiments. Stained sections from in vivo tumor samples were imaged on a Perkin Elmer Vectra 3.0 with spectral unmixing of images performed using Perkin Elmer Inform software. Image quantification was performed using Cell Profiler v.3 as described in the Results .
4
ZIKV EDIII-specific Antibody Analysis
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Mouse sera were analyzed for ZIKV EDIII-specific antibody responses by ELISA as previously described32 (link),35 (link). Briefly, ELISA plates were coated with ZIKV EDIII-His or E1-454 protein overnight at 4 °C and blocked with 2% fat-free milk for 2 h at 37 °C. Serially diluted mouse sera were added to the plates and then incubated for 1 h at 37 °C. After washing, the plates were incubated with HRP-conjugated anti-mouse IgG (1:5000), anti-mouse IgG1 (1:2000, Bethyl Laboratories, Montgomery, TX, USA), or anti-mouse IgG2a (1:2000, Invitrogen) for 1 h at 37 °C. The reaction was detected by 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Invitrogen) and stopped by 1 N H2SO4. The absorbance at 450 nm was measured using an ELISA plate reader (Tecan, Morrisville, NC, USA), and the respective antibody titers were calculated.
5
Immunohistochemical Analysis of Chicken Hindgut
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A hindgut was dissected from chicken embryo and fixed in 4% (w/v) paraformaldehyde (PFA)/phosphate buffered saline (PBS: 0.14 M NaCl, 2.7 mM KCl, 10 mM Na2HPO4–12H2O, 1.8 mM KH2PO4) for 10 min at room temperature (RT). The specimen was washed in PBS twice for 5 min each at RT, and embedded in FSC 22 Clear frozen section compound (Leica, 3801480). Cryostat sections of 20 μm were prepared (Thermo Scientific, Cryostar NX70). Following drying on the hotplate at 37°C, the sections were re-fixed in 4% PFA for 5 min at RT. After washing three times in PBS for 5 min each at RT, the sections were incubated with 0.5% blocking reagent (Roche, 1096176)/PBS for 1 h at RT, followed by primary antibodies; 1:300 dilution of anti-αSMA (abcam, ab5694) and 1:300 dilution of Tuj1 (R & D systems, MAB1195) overnight at 4°C. Following three times washing in PBS for 5 min each at RT, they were incubated with Alexa 488-conjugated second antibodies; 1:300 dilution of anti-rabbit IgG (H + L) (donkey; Invitrogen, A21206), 1:300 dilution of anti-mouse IgG2a (goat; Invitrogen, A21131) and 1:2000 dilution of DAPI (Nacalai Tesque, 11034–56) for 1.5 h at RT. After washing three times for 10 min each at RT, the specimens were sealed with Fluoromount (Diagnostic BioSystems). Fluorescent images were obtained using a Nikon A1R confocal microscope.
6
Quantification of SARS-CoV-2 Antibody Responses
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Enzyme-linked immunoassay (ELISA) was used to measure specific serum antibodies from immunized mice.15 (link),26 (link) Specifically, 96-well ELISA plates were precoated with each recombinant SARS-CoV-2 S or RBD protein (1 μg/mL)26 (link) at 4°C overnight, and blocked with blocking buffer (e.g., 2% fat-free milk dissolved in PBST (0.05% Tween-20 in PBS)) at 37°C for 1 h. The plates were then incubated with serially diluted mouse sera at 37°C for 1 h, and washed with PBST for at least three times. This step was followed by further incubation of the plates with horseradish peroxidase (HRP)-conjugated anti-mouse IgG-Fab (1:5,000, Sigma), anti-mouse IgG1, anti-mouse IgG2a, and anti-mouse IgG2b (1:5,000, Invitrogen) antibodies, respectively, at 37°C for 1 h, and washing for three times. After incubation of the plates with 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate (Sigma), the reaction was stopped by addition of 1 N H2SO4. The absorbance at 450 nm was measured using Cytation 7 Microplate Multi-Mode Reader and Gen5 software (BioTek Instruments).
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