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
Q9BZS1 Primary Antibody FOXP3 Human Mouse IgG1 236A/E7 Thermo Fisher Scientific 41-4777-82 eF570 AB_2573609 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 [3, 1] NA 0000-0003-4379-8967  
Q99JB6 Primary Antibody FOXP3 Mouse Rat IgG2a FJK-16s Thermo Fisher Scientific 41-5773-82 eF570 AB_11219073 Stock IBEX2D Manual 1:4 Cytofix/Cytoperm Fixed Frozen Small Intestine NA 0.3% Triton-X-100 NA 1 mg/ml LiBH4 15 minutes Yes 0000-0003-4379-8967 [3] NA 0000-0003-4379-8967 1
Q9BZS1 Primary Antibody FOXP3 Human Mouse IgG1 236A/E7 Thermo Fisher Scientific 41-4777-82 eF570 AB_2573609 Stock IBEX2D Manual 1:4 Cytofix/Cytoperm Fixed Frozen Lymph Node Follicular Lymphoma 0.3% Triton-X-100 NA 1 mg/ml LiBH4 15 minutes Yes 0000-0003-4379-8967 [2] NA 0000-0003-4379-8967  
Q9BZS1 Primary Antibody FOXP3 Human Mouse IgG1 236A/E7 Thermo Fisher Scientific 41-4777-82 eF570 AB_2573609 Stock Multiplexed 2D Imaging FFPE Tonsil NA 0.3% Triton-X-100 pH 6 for 30 minutes ER1 (AR9961) and pH 9 for 30 minutes ER2 (AR9640) using the Leica Bond NA No 0000-0003-4379-8967 NA 0000-0003-4379-8967 2,3

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., “A Multi-scale, Multiomic Atlas of Human Normal and Follicular Lymphoma Lymph Nodes”, bioRxiv, 2022, doi: 10.1101/2022.06.03.494716.

  3. 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. Validated by Evelyn Kandov (No ORCID)
  2. This antibody works well in fixed frozen tissues but is very dim in human FFPE samples. This conjugate is particularly challenging due to autofluorescence from red blood cells. Red blood cells are small and round like the anticipated nuclear pattern of FOXP3. FOXP3 needs to be amplified with a secondary antibody in human FFPE samples. Clone also appears to be sensitive to antigen retrieval conditions (pH 9 is best).
  3. Antibody labels nuclei of T cells in FFPE tissues after dual antigen retrieval (pH 6 and pH 9). Signal is dim and the eF570 conjugate can give a false positive with autofluorescence from red blood cells. Recommendations: Amplify signal with a secondary antibody, perform dual antigen retrieval, and use at a concentration of 1:25 for best signal.
Human tonsil FFPE : FOXP3 (yellow, catalog number 41-4777-82) and Hoechst (blue, catalog number 40046)
Human lymph node: CD25 (cyan, catalog number 356128) and FOXP3 (yellow, catalog number 41-4777-82)