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
P07766 Primary Antibody CD3 Human Mouse IgG1 UCHT1 BioLegend 300402 (Unconjugated) AF555 NA Custom 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
P07766 Primary Antibody CD3 Human Rabbit IgG EP4426 Abcam ab208514 AF555 AB_443425 Stock IBEX2D Manual FFPE Tonsil NA 0.3% Triton-X-100 pH 6 for 40 minutes at 95C (AR6 Akoya Biosciences AR600250ML) 1 mg/ml LiBH4 15 minutes Yes 0000-0003-4379-8967 NA 0000-0003-4379-8967 2
P04234 Primary Antibody CD3 Human Mouse IgG1 UCHT1 BioLegend 300401 (Unconjugated) AF555 NA Custom IBEX2D Manual 1:4 Cytofix/Cytoperm Fixed Frozen Lymph Node NA 0.3% Triton-X-100 NA NA Yes 0000-0003-4379-8967 NA 0000-0003-4379-8967 3
P07766 Primary Antibody CD3 Human Rabbit IgG EP4426 Abcam ab208514 AF555 AB_443425 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 Yes 0000-0003-4379-8967, 0009-0006-9784-2694 NA 0000-0003-4379-8967 2
P07766 Primary Antibody CD3 Human Rabbit IgG EP4426 Abcam ab208514 AF555 AB_443425 Stock Cell DIVE-IBEX 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 1 mg/ml LiBH4 15 minutes Yes 0000-0003-4379-8967 NA 0000-0003-4379-8967 4
P07766 Primary Antibody CD3 Human Rabbit IgG EP4426 Abcam ab208514 AF555 AB_443425 Stock Cell DIVE-IBEX FFPE Lung Cancer 0.3% Triton-X-100 pH 6 for 30 minutes ER1 (AR9961) and pH 9 for 30 minutes ER2 (AR9640) using the Leica Bond 1 mg/ml LiBH4 15 minutes Yes 0000-0003-4379-8967 NA 0000-0003-4379-8967 5
P07766 Primary Antibody CD3 Human Rabbit IgG EP4426 Abcam ab208514 AF555 AB_443425 Stock Cell DIVE-IBEX FFPE Lymph Node Follicular Lymphoma 0.3% Triton-X-100 pH 6 for 30 minutes ER1 (AR9961) and pH 9 for 30 minutes ER2 (AR9640) using the Leica Bond 1 mg/ml LiBH4 15 minutes Yes 0000-0003-4379-8967 [3] NA 0000-0003-4379-8967  
P07766 Primary Antibody CD3 Human Rabbit IgG EP4426 Abcam ab208514 AF555 AB_443425 Stock Cell DIVE-IBEX FFPE Lymph Node 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 1 mg/ml LiBH4 15 minutes Yes 0000-0003-4379-8967 [3] NA 0000-0003-4379-8967  

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

  3. 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.

Additional Notes

  1. Clone UCHT1 labels T cells in fixed frozen human tissues. Several conjugates are suitable for Multiplexed 2D Imaging; however, we recommend the following conjugates for IBEX experiments: AF488, AF555, PE, iF594, and AF647. Custom conjugation from BioLegend.
  2. Evaluated in human tonsil FFPE samples with CD20 (clone L26) and Hoechst (nuclear marker). Antibody labels T cells. Preferred clone for FFPE samples.
  3. Custom conjugate by BioLegend. Labels T cells in diverse tissues.
  4. Antibody labels T cells. Preferred clone for FFPE samples. Works beautifully on Cell DIVE imager with IBEX dye inactivation protocol.
  5. Labels T cells in lung. Great signal and works very well on Cell DIVE imager with IBEX dye inactivation protocol.
Human normal LN: BCL2 (cyan, catalog number ab236221 and A-31573), CD3 (magenta, catalog number ab208514), CD4 (blue, catalog number ab196372), CD21 (yellow, catalog number ab306325)
Human FL LN: BCL2 (cyan, catalog number ab236221 and A-31573), CD3 (magenta, catalog number ab208514), CD4 (blue, catalog number ab196372), CD21 (yellow, catalog number ab306325)