Immunohistochemical Analysis of Siglec-F Ligands

Siglec-F ligand expression was assessed as described [12] (link), with minor modifications. 8 μm sections were blocked for 1 h with 5% normal sera from mouse and goat (Sigma) diluted in 0.5% casein solution (Vector Labs, Peterborough, UK). 1 μg/ml siglec-F-Fc (in house) was pre-complexed to anti-human Fc Alexa-488 (Jackson ImmunoResearch, Newmarket, UK) at a ratio of 1:1 for 1 h 4 °C prior to incubation on slides for 24 h 4 °C. To evaluate background fluorescence, sections were stained with an irrelevant Fc-protein. To evaluate sialic acid dependent binding, sections were treated with 0.17 U/mL Vibrio cholerae sialidase (Sigma) in HBSS at 37 °C for 1 h prior to block. All sections were incubated with DAPI (Vector Labs) at 0.5 μg/mL in PBS for 5 min to label nuclei and confocal images were acquired (LSM 700 microscope and Zen 2009 software; Carl Zeiss). Multi-channel images were created and processed in parallel using Photoshop software (Adobe).