Journal: Nature protocols

Article Title: Qualifying antibodies for image-based immune profiling and multiplexed tissue imaging

doi: 10.1038/s41596-019-0206-y

Figure Lengend Snippet: t-CyCIF staining plan for our immune profiling panel

Article Snippet: For some antigens, stereotypical variability is also observed in each tissue (e.g., CD markers on T cells) and this can serve as an internal staining control. table ft1 table-wrap mode="anchored" t5 Table 2 | caption a7 Cycle 0 488/FITC Dil. 555/Cy3 Dil. 1000 647/Cy5 Name Clone Vendor Cat. no. Name Goat antirabbit Clone Vendor Thermo Fisher Cat. no. A-21428 Name Goat antimouse Clone Vendor Thermo Fisher Cat. no. A-21235 Dil. 1000 1 LAG3 EPR4392(2) Abcam ab180187 100 2 Ki-67 D3B5 CST 11882s 100 Keratin AE1/AE3 eBioscience 41-9003-80 200 PD-1 EPR4877(2) Abcam ab 201825 100 3 CD45RB PD7/26 eBioscience 53-9458-80 100 CD3 EP4426 Abcam ab208514 100 PD-L1 E1L3N CST 15005S 50 CD4 Polyclonal R&D Systems FAB8165G 100 CD45 2D1 R&D Systems FAB1430P-025 100 CD8a AMC908 eBioscience 50-0008-80 100 5 CD163 EPR14643 Abcam ab218293 100 CD68 D4B9C CST #79594 100 CD14 EPR3653 Abcam ab196169 100 6 CD11b C67F154 eBioscience 53-0196-80 100 Foxp3 236A/E7 eBioscience 41-4777-80 100 7 IBA1 EPR6136 Abcam ab195031 250 α-SMA EPR5368 Abcam ab202509 250 CD20 L26 eBioscience 50-0202-80 250 8 CD19 EPR5906 Abcam ab196468 100 GFAP GA5 eBioscience 41-9892-80 100 Open in a separate window t-CyCIF staining plan for our immune profiling panel Considerations in the construction of antibody panels Even when individual antibodies have been validated and the guidelines described in Box 2 considered, it is necessary to demonstrate that mixtures of antibodies perform as expected.

Techniques: Staining

Journal: Nature protocols

Article Title: Qualifying antibodies for image-based immune profiling and multiplexed tissue imaging

doi: 10.1038/s41596-019-0206-y

Figure Lengend Snippet: a, All PD-1+ cells (n = 273) from LUNG-3-PR were further separated using binary gating of all 15 immune markers. 21 putative subpopulations comprising three or more cells are shown. Cluster 3 is the subpopulation with PD-1+/LAG3+/CD8a+/CD3+ (described by visual review of scatter plots in Fig. 7). Images of all 125 cells binned into putative rare cell clusters 1 through 21 are shown in Supplementary Figs. 17–22. Supplementary Table 10 indicates whether visual review of the images by a trained pathologist was deemed to be consistent or inconsistent with the automated calls. Nine of the 21 clusters (1–4, 6, 8, 10, 11, and 17) were validated by visual review (38 of 125 individual rare cell calls). b, A gallery of images of individual markers from single cells from clusters 1 (CD45+/CD45RB+/CD3+/CD8a+/PD1+), 2 (CD45+/CD45RB+/CD3+/CD4+/PD1+), and 3 (CD45+/CD45RB+/CD3+/CD8a+/PD1+/LAG3+) are shown (with the exception of CD45RB). Images are shown for representative cells from each of these three clusters (scale bar, 25 μm).

Article Snippet: For some antigens, stereotypical variability is also observed in each tissue (e.g., CD markers on T cells) and this can serve as an internal staining control. table ft1 table-wrap mode="anchored" t5 Table 2 | caption a7 Cycle 0 488/FITC Dil. 555/Cy3 Dil. 1000 647/Cy5 Name Clone Vendor Cat. no. Name Goat antirabbit Clone Vendor Thermo Fisher Cat. no. A-21428 Name Goat antimouse Clone Vendor Thermo Fisher Cat. no. A-21235 Dil. 1000 1 LAG3 EPR4392(2) Abcam ab180187 100 2 Ki-67 D3B5 CST 11882s 100 Keratin AE1/AE3 eBioscience 41-9003-80 200 PD-1 EPR4877(2) Abcam ab 201825 100 3 CD45RB PD7/26 eBioscience 53-9458-80 100 CD3 EP4426 Abcam ab208514 100 PD-L1 E1L3N CST 15005S 50 CD4 Polyclonal R&D Systems FAB8165G 100 CD45 2D1 R&D Systems FAB1430P-025 100 CD8a AMC908 eBioscience 50-0008-80 100 5 CD163 EPR14643 Abcam ab218293 100 CD68 D4B9C CST #79594 100 CD14 EPR3653 Abcam ab196169 100 6 CD11b C67F154 eBioscience 53-0196-80 100 Foxp3 236A/E7 eBioscience 41-4777-80 100 7 IBA1 EPR6136 Abcam ab195031 250 α-SMA EPR5368 Abcam ab202509 250 CD20 L26 eBioscience 50-0202-80 250 8 CD19 EPR5906 Abcam ab196468 100 GFAP GA5 eBioscience 41-9892-80 100 Open in a separate window t-CyCIF staining plan for our immune profiling panel Considerations in the construction of antibody panels Even when individual antibodies have been validated and the guidelines described in Box 2 considered, it is necessary to demonstrate that mixtures of antibodies perform as expected.

Techniques: