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. 2023 Jan 23;4(2):222–239. doi: 10.1038/s43018-022-00506-7

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — a, Schematic of L-PLA using fucosylated HLA-DRB1 (fuco-HLA-DRB1) as an example. We stained for (1) HLA-DRB1 using anti-HLA-DRB1 antibody followed by oligonucleotide-conjugated PLA secondary antibody (2° Ab) (+) and (2) fucosylated glycan using biotinylated (‘B’) AAL lectin followed by anti-biotin antibody followed by oligonucleotide-conjugated PLA secondary (−). Ligated PLA oligonucleotides were subjected to rolling circle amplification PCR (RCA PCR), giving rise to fluorescent punctae. b, Representative images of secondary antibody-only control (top) or full L-PLA (bottom) staining of endogenous, fucosylated HLA-DRB1 (green) performed on coverslip-grown WM793 cells (with phalloidin (red) and DAPI (blue) co-stains). c, To further demonstrate that fucosylated HLA-DRB1 L-PLA staining is fucosylation species-specific, we performed L-PLA of endogenous, fucosylated HLA-DRB1 (green) on WM793 cells treated with DMSO or FUTi (phalloidin (red) and DAPI (blue) co-stains). d, To demonstrate specificity of individual L-PLA primary antibodies, FFPE melanoma tissue was stained for a melanoma marker (MART1 and S100 cocktail; red), AAL–FITC (green), HLA-DRB1 (white) and DAPI (blue). e, Representative images of secondary antibody-only control (top) or full L-PLA (bottom) staining of endogenous, fucosylated HLA-DRB1 (green) performed on human melanoma specimens (with MART1 and S100 (red) and DAPI (blue) co-stains). f, FFPE melanoma tissues were subjected to L-PLA HLA-DRB1 staining with or without washing with 500 mM l-fuc and subsequent staining with MART1 and S100 (red) and DAPI (blue). Total loss of fucosylated HLA-DRB1 (green) signal in tissue washed with l-fuc confirms the fucose-specificity of L-PLA for fucosylated HLA-DRB1. Single melanoma marker and fucosylated HLA-DRB1 channels are shown in white for clear visualization. For b–f, representative images are shown for n = 3 independent biological replicate experiments.

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