For mouse T cell and B cell analysis in lymphoid tissues, LN cells were stained for viability and extracellular antigens with directly labeled antibodies, CD95 (BioLegend 152606), IgD (BioLegend 405710), GL7 (BioLegend 144617), live/dead fixable yellow dead cell staining (Thermo Fisher Scientific L34968), CD19 (BioLegend 115555), biotinylated SARS-CoV-2-S1 protein (BioLegend 793804), anti-biotin (BioLegend 409003), CD3 (BioLegend 100220), CD3 (BioLegend 100204), CD4 (BioLegend 100540), ICOS (BioLegend 117420), CD8 (BioLegend 557654), CD45 (BioLegend 103134), CXCR5 (BioLegend 145504), PD-1 (BioLegend 135231), CD38 (BioLegend 102712), IgG1 (BioLegend 406620), and IgG2a (BioLegend 407114). Cells were washed, fixed, and permeabilized by using the Ebioscience Foxp3/Transcription Factor Staining Buffer Set (Thermo Scientific 00-5523-00) kit according to manufacture instructions. Permeabilized cells were intracellularly stained with Bcl-6 (BioLegend 358512). Cells were acquired on an Attune NxT Flow Cytometer and analyzed with Attune NxT software v.4.2.0.
Anti biotin
Manufactured by BioLegend
Anti-biotin is a laboratory reagent used to bind and neutralize biotin in biological samples. It is designed to prevent interference from biotin in immunoassays and other applications where biotin may be present.
Automatically generated - may contain errors
2 protocols using anti biotin
1
Multicolor Flow Cytometry for Mouse Lymphoid Tissue Analysis
2
Multiplex RNA and Protein Detection
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Slides were processed as above for RNAscope protocol using DNASE1L3 (ACDbio, #576121-C1), ITGAX (ACDbio, #419151-C2) and Eubacteria 16S (#464461-C3) probes. C1 oligos were developed with TSA-Digoxygenin (Akoya Biosciences, NEL748001KT), C2 oligos with TSA-FITC, (Akoya Biosciences, #NEL741001KT) and C3 oligos with TSA-Biotin (Akoya Biosciences, NEL749A001KT) diluted 1:100 in TSA buffer. After the RNAscope protocol, slides were washed, blocked, and stained using Panel 2 and 3 (Supplementary Table 2 ) as described in the IMC paragraph above. Panels 2 and 3 incorporated anti-Digoxygenin (RD System, #611621), anti-FITC (Standard BioTools, #3174006B) and anti-Biotin (Biolegend, #409002) metal-tagged antibodies to detect the RNAscope staining output.
For quantification of co-expression of DNASE1L3 and Lysozyme or DNASE1L3 and NOX2 expressing cells, DNASE1L3+ cells were segmented using the FindPrimaryObjects module in CellProfiler using the DNASE1L3 channel as input72 (link). The expression of all markers was calculated for each cell and arcsinh transformed, and cells were manually labelled as belonging to the SED using a custom R script.
For quantification of co-expression of DNASE1L3 and Lysozyme or DNASE1L3 and NOX2 expressing cells, DNASE1L3+ cells were segmented using the FindPrimaryObjects module in CellProfiler using the DNASE1L3 channel as input72 (link). The expression of all markers was calculated for each cell and arcsinh transformed, and cells were manually labelled as belonging to the SED using a custom R script.
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