Single-cell suspensions from the bladders and spleens of sacrificed mice were obtained as previously described [39 (link),40 (link)]. Briefly, splenocytes were obtained by mechanical dissociation, while bladders were minced and digested step-wise with 0.5 mg/mL thermolysin (Roche, Basel, Switzerland) and 1 mg/mL collagenase/dispase (Roche). IFN-γ ELISPOT assays were performed as described [39 (link)] using Multiscreen-HA 96-well plates (MAHA S4510, Millipore, Billerica, MA, USA), anti-IFN-γ monoclonal antibody (R4-6A2, Beckton Dickinson Pharmingen), biotinylated anti-IFN-γ monoclonal antibody (XMG1.2, Beckton Dickinson Pharmingen) and Streptavidin-AP (Roche). 100,000 splenocytes/well in duplicates were incubated with 0.5 μg/mL of H-2Db restricted Uty246–254 peptide or medium alone (control wells) for 16–24 h. Uty-specific responses were defined as the number of IFN-γ spots/105 cells in the Uty-stimulated wells minus the number of IFN-γ spots/105 cells in the control wells (<3 spots/well). T cell staining was performed as previously described [41 (link)], using PE-conjugated Uty246–254 H-2Db-restricted dextramers (Immudex, København, Denmark) and APC-labeled CD8α (clone 53-6.7, eBioscience, San Diego, CA, USA). Flow-cytometer cell acquisition and analysis were performed as described above.
Streptavidin ap
Manufactured by Roche
Sourced in Switzerland
Streptavidin-AP is a laboratory reagent used for detection and quantification in various bioanalytical techniques. It is composed of streptavidin, a protein with a high affinity for biotin, conjugated to alkaline phosphatase, an enzyme that catalyzes a colorimetric reaction. This product can be utilized in applications such as ELISA, Western blotting, and other bioassays that involve the biotin-streptavidin interaction.
Automatically generated - may contain errors
Lab products found in correlation
Tris glycine, by Thermo Fisher Scientific (2 mentions)
Nucleic acid blocking reagent, by Roche (2 mentions)
Cdp star, by Roche (2 mentions)
Multiscreen ha 96 well plates, by Merck Group (1 mentions)
Collagenase dispase, by Roche (1 mentions)
Thermolysin, by Roche (1 mentions)
Envision multilabel reader, by PerkinElmer (1 mentions)
Developing buffer, by Thermo Fisher Scientific (1 mentions)
Anti igm biotin, by Southern Biotech (1 mentions)
Anti igg1 biotin, by Southern Biotech (1 mentions)
Flexstation 3, by Molecular Devices (1 mentions)
Blocking reagent, by Roche (1 mentions)
Biospectrum imaging system, by Analytik Jena (1 mentions)
Anti dig ap, by Roche (1 mentions)
Rmm 1, by BioLegend (1 mentions)
Tnp bsa, by Biosearch Technologies (1 mentions)
Antibody diluent, by Agilent Technologies (1 mentions)
Eukitt, by Merck Group (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
8 protocols using streptavidin ap
1
Isolation and Analysis of Murine Immune Cells
2
Quantification of CD9 in Exosomes
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The CD9 level in exosomes was determined by a sandwich ELISA. A MaxiSorp micro titer plate (Thermo Fisher Scientific, MA, USA) was coated with 5 μg/mL anti-CD9 antibody (Ancell Corporation, Bayport, MN, USA) in carbonate buffer (pH 9.6) at 4 °C overnight. After washing 3 times with PBS, 200 μL of 1% BSA/PBS was added and incubated at room temperature for 1 h with shaking. After washing, sample was added in a final volume of 100 μL and incubated at room temperature for 2 h. After washing, 0.5 μg/mL biotinylated anti-CD9 antibody (Ancell Corporation) in 1% BSA/PBS was added in a final volume of 100 μL and incubated at room temperature for 1 h. After washing, 1:5000 diluted streptavidin-AP (Roche, Basel, Switzerland) in 1% BSA/PBS was added in a final volume of 100 μL and incubated at room temperature for 1 h. After washing 6 times with PBS, CDP-Star substrate with Emerald II Enhancer (Thermo Fisher Scientific) was added and chemiluminescence was recorded by the EnVision Multilabel Reader (PerkinElmer, MA, USA).
3
Quantifying TBP Binding Complexes
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Briefly, as previously described, we obtained nuclear extract of sgNSUN6 and non-targeting controls edited cells as described18 , 19 (link) and incubated it with the TBP probe labeled with biotin on the 5ʹ and 3ʹ end as described16 (link) and followed protocol described. To inhibit the TBP complexes, we treated the nuclear extracts with Sarkosyl, an inhibitor of NELFB. We then resolved the complexes formed in 4%–20% Tris Glycine (Novex) and blotted it into a hybond N+ membrane. The membrane was air dried and then, blocked with a nucleic acid blocking reagent (Roche) in PBS for 1 h, then incubated with 1:1000 Streptavidin AP (Roche) for 2 h, washed with PBS, and then imaged with CDP star (Roche) in a biorad chemidoc imaging system.
4
Quantifying IgG1 and IgM Antibodies in Mice
5
Microarray Hybridization and Signal Detection
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Hybridization of amplicons to probes on microarrays was performed according to a previously described protocol (Hsiao et al., 2005 ), with modifications. Briefly, 4 μl of labelled amplicons were added to 220 μl of hybridization solution [5 × SSC (v/v), 2% blocking reagent (w/v; Roche Applied Science), 0.1% N‐lauroylsarcosine (w/v) and 0.02% SDS (w/v)], denatured with boiling water for 7 min, and immediately chilled on ice for 10 min. Hybridization was conducted at 58°C for 40 min, and arrays were then washed twice at 56°C for 5 min each with 0.25 × SSC to remove non‐hybridized PCR products, and incubated for 30 min with 200 μl of blocking solution [1% (wt/vol) blocking reagent dissolved in maleic acid buffer (0.1 M maleic acid, 0.15 M NaCl, pH 7.5)] containing either anti‐DIG‐AP (1:5,000 dilution; Roche Applied Science) or streptavidin‐AP (1:1,000 dilution; Roche Applied Science). Signals were colour developed with 500 μl nitroblue tetrazolium (NBT)/5‐bromo‐6‐chloro‐3‐indolyl phosphate, p‐toluidine salt (BCIP) solution (Roche Applied Science) at room temperature without shaking. PVC chip signals were captured and analysed with Dr AiM reader (Dr Chip Biotechnology), while nylon membrane signals were captured with a BioSpectrum Imaging System (UVP, Upland, CA, USA) and analysed with Vision Works LS Analysis Software v6.5.2 (UVP).
6
ELISA for Anti-TNP IgG3/IgM Antibodies
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High‐binding 384‐well plates (Grenier Bio‐one, Kremsmünster, Austria) were coated overnight with TNP‐BSA (Biosearch Technologies) at 10 μg/ml. Plates were washed between each step with PBS containing 0·05% Tween. Blocking was performed using 4% BSA. Serum was diluted 1 in 50 in PBS with 1% BSA. The secondary antibodies anti‐IgG3 (R40‐82, BD Biosciences) or anti‐IgM (RMM‐1, BioLegend), were then applied in a solution of 1% skimmed milk and 1% BSA. Streptavidin‐AP (Roche, Basel, Switzerland) was added in 0·1% BSA. Finally p‐nitophenyl phosphate (Sigma‐Aldrich, St Louis, MO) at 1 mg/ml in NPP buffer was applied and plates were read at 405 nm using a Biotek plate reader (Biotek, Winooski, VT).
7
Immunohistochemical Staining of Paraffin-Embedded Tissues
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Paraffin-embedded tissues were stained immunohistochemically. Tissues were cut in 5 mm serial sections. Antigen retrieval was performed in an acidic citrate buffer for protein gene product 9.5 (PGP9.5) and substance P (SP) or an alkaline target retrieval buffer for NGF, neurotrophin 3 (NT-3), and brainderived neurotrophic factor (BDNF). Primary and biotinylated secondary antibodies (Table 1) and streptavidin-AP (Roche Diagnostics, Rotkreuz, Switzerland) were diluted in antibody diluent (DakoCytomation, Glostrup, Denmark). The antibodies were incubated at room temperature with individual dilution factors and incubation times ranging from 40 to 75 minutes or overnight at 4 C. Antibody-antigen detection was performed using the Fast Red Substrate System (Thermo Fisher Scientific, Waltham, USA). Sections were counterstained with hematoxylin (Merck KGaA, Darmstadt, Germany) and mounted with Eukitt (Sigma-Aldrich, St. Louis, USA).
8
Inhibition of TBP Complexes Assay
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Briefly as previously described (14) . We obtained nuclear extract of sgNSUN6 and non-targeting controls edited cells as described (16, 17) and incubated it with TBP probe labelled with biotin on 5' and 3' end as described (19) and followed protocol described (14) . To inhibit, the TBP complexes, we treated the nuclear extracts with Sarkosyl, an inhibitor of NELFB. We then resolved the resolved the complexes formed in 4-20% Tris Glycine (Novex) and blotted it unto a hybond N+ membrane. The membrane was air dried and then blocked with nucleic acid blocking reagent (Roche) in PBS for 1hr and then incubated with 1:1000 Streptavidin AP (Roche) for 2hrs and then washed with PBS and then imaged with CDP star (Roche) in a biorad chemidoc imaging system.
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