Mouse igg2b isotype control

The ICC staining was performed using cytospin slides prepared with freshly isolated BAL cells harvested from LTRs. These cytospin samples were air-dried and frozen (− 80 °C) until further processing. For study processing, the cytospin slides were thawed and fixed in formaldehyde (4%). To reduce unspecific staining, the slides were incubated first with protein serum-free block (Dako® Agilent Technologies, Denmark), followed by horse serum (5%), and, subsequently, by BLOXALL (Vector Laboratories® Ca, USA). After this, the slides were incubated with primary monoclonal mouse anti-human IL-26 antibodies (5 ug/ml) (Clone 197505, R&D Systems Inc., Mi, USA) or mouse IgG2b isotype control (Clone 20116, R&D Systems Inc.). After washing, slides were incubated with a secondary antibody from the anti-mouse immPRESS kit (5ug/l) (Vector Laboratories®). Bound antibodies were then visualized by ImmPACT VIP-substrate chromogen system (Vector Laboratories®). The procedure was then repeated with in-between washes, but with a primary rabbit anti-human CD68 antibody (5ug/ml) (Abbiotech® Ca, USA), followed by a secondary antibody from the anti-rabbit immPRESS kit (Vector Laboratories®). Bound antibodies were now visualized by ImmPACT DAB-substrate chromogen system (Vector Laboratories®), and slides were finally counterstained with Methyl Green (Vector Laboratories®).

Cells were stained with mouse IgG_2B isotype control (R&D Systems, Minneapolis, MI, USA) or primary mouse anti-KIR3DL3 antibody kindly provided by Professor Ashley Moffet, University of Cambridge, UK [16 (link)]. Where necessary, a goat anti-mouse PE secondary antibody was used (R&D Systems, Minneapolis, MI, USA). The level of KIR3DL3 expression was analyzed by the Beckman Coulter Gallios and the Kaluza Flow Analysis Software (Beckman Coulter, Fullerton, CA, USA). Percentage of positive cells and mean fluorescence intensity (MFI) were calculated following consideration of the values from the isotype control.