UniProt Accession Number Reagent Type Target Name / Protein Biomarker Target Species Host Organism Isotype Clonality Vendor Catalog Number Conjugate RRID Availability Method Tissue Preservation Target Tissue Tissue State Detergent Antigen Retrieval Conditions Dye Inactivation Conditions Recommend Agree Disagree Contributor Notes
P20023 Primary Antibody CD21 Human Mouse IgG1 Bu32 BioLegend 354903 PE AB_2561406 Stock IBEX2D Manual 1:4 Cytofix/Cytoperm Fixed Frozen Lymph Node NA 0.3% Triton-X-100 NA 1 mg/ml LiBH4 15 minutes Yes 0000-0003-4379-8967 [2, 1] NA 0000-0003-4379-8967 1
P20023 Primary Antibody CD21 Human Rabbit IgG SP186 Abcam ab306325 PE NA Stock Cell DIVE-IBEX FFPE Lymph Node Follicular Lymphoma 0.3% Triton-X-100 pH 6 for 30 minutes ER1 (AF9961) and pH 9 for 30 minutes ER2 (AF9640) using the Leica Bond 1 mg/ml LiBH4 15 minutes Yes 0000-0003-4379-8967 NA 0000-0003-4379-8967 2
P20023 Primary Antibody CD21 Human Rabbit IgG SP186 Abcam ab306325 PE NA Stock Cell DIVE-IBEX FFPE Lymph Node Follicular Lymphoma 0.3% Triton-X-100 pH 6 for 30 minutes ER1 (AF9961) using the Leica Bond 1 mg/ml LiBH4 15 minutes No 0000-0003-4379-8967 NA 0000-0003-4379-8967 2

Publications

  1. A. J. Radtke et al., “IBEX: an iterative immunolabeling and chemical bleaching method for high-content imaging of diverse tissues”, Nat. Protoc., 17(2):378-401, 2022, doi: 10.1038/s41596-021-00644-9.

    A. J. Radtke et al., “Accompanying dataset for: IBEX: An iterative immunolabeling and chemical bleaching method for high-content imaging of diverse tissues”, doi: 10.5281/zenodo.5244550.

  2. A. J. Radtke et al., “IBEX: A versatile multiplex optical imaging approach for deep phenotyping and spatial analysis of cells in complex tissues”, Proc Natl Acad Sci, 117(52):33455–33465, 2020, doi:10.1073/pnas.2018488117

Additional Notes

  1. Evaluated in human lymph nodes and shown to label follicular dendritic cells and B cells. Clone Bu32 has been shown to work in multiple conjugates for IBEX experiments (AF488 and AF532).

  2. Clone SP186 labels follicular dendritic cells in FFPE samples following antigen retrieval with pH 6 and amplification with a secondary antibody. This direct conjugate gives beautiful labeling after dual antigen retrieval (pH 6 and pH 9) but a speckled, less robust labeling pattern with pH 6 alone (shown in the two images below).

    Antigen retrieval, pH 6 and pH 9 Antigen retrieval, pH 6
    CD21 PE FL pH6and9 CD21 PE FL pH6