The double immunofluorescence antigen labelling of frozen human lung sections was performed with LeA, LeX, SLeA and SLeX, with EpCAM antibody. The frozen human lung sections were fixed in Acetone/Ethanol (75:25) for 10 min and washed twice with TBS. The sections were blocked with 5% normal donkey serum (Jackson ImmunoResearch Lab Inc, West Grove PA) for an hour at room temperature. The sections were incubated with rabbit anti-EpCAM (1:300, Abcam, Cat#: ab32392) plus (1) FITC conjugated mouse anti-LeA (1:100, Santa Cruz, cat#: sc-51512) and (2) FITC conjugated mouse anti-human CD15 (LeX, 1:50); (3) mouse anti-SLeA (1:500, CA19.9) and (4) Rat anti-SLeX (1:200, BD 555946) overnight at 4 °C. The slides were washed three times in TBS and incubated with Alexa 647 conjugated donkey anti-rabbit secondary antibodies (Jackson ImmunoResearch Lab, 1:300) for (1) and (2) and CY3 conjugated donkey anti-mouse (Jackson ImmunoResearch Lab, 1:300) and Alexa 647 conjugated donkey anti-rabbit secondary antibodies (Jackson ImmunoResearch Lab, 1:300) for (3) and CY3 conjugated donkey anti-rat (Jackson ImmunoResearch Lab, 1:300) and Alexa 647 conjugated donkey anti-rabbit secondary antibodies (Jackson ImmunoResearch Lab, 1:300) for (4). Samples were then washed three times with TBS and mounted with Prolong Gold anti-fade mounting media containing DAPI (Invitrogen).
Jia N., Byrd-Leotis L., Matsumoto Y., Gao C., Wein A.N., Lobby J.L., Kohlmeier J.E., Steinhauer D.A, & Cummings R.D. (2020). The Human Lung Glycome Reveals Novel Glycan Ligands for Influenza A Virus. Scientific Reports, 10, 5320.
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