Fitc conjugated anti goat igg

Non-immune IgM and α₂M binding to IEs was detected by flow cytometry as previously described [24 (link), 26 (link), 32 (link)]. Binding of both proteins at 10 nM has been detected in several laboratory clones [23 (link)–26 (link)], but included two additional concentrations (1 nM and 100 nM) to explore the potential binding in the field isolates. Briefly, intact and unfixed late trophozoite stage IEs purified by magnet-activated cell sorting (MACS) were incubated with either 1, 10, or 100 nM non-immune human IgM or α₂M in PBS supplemented with 1% Ig-free bovine serum albumin (PBS 1% BSA). IE-bound IgM was measured with a FITC-conjugated anti-human IgM (1:150; Sigma), whereas α₂M was determined with a combination of goat polyclonal anti-human α₂M (1:300; Abcam, UK) and FITC-conjugated anti-goat IgG (1:150; Vector, UK). Non-immune IgM and α₂M binding to IEs was quantified as the median fluorescence intensity (MFI) in IEs labelled with 10 µg/mL Hoechst. BD LSR II flow cytometer was used for data acquisition and FlowLogic software (Inivai Technologies, Australia) for data analysis.

Stomachs from 30- to 90-day-old Bmpr1a^ΔMES and control littermates were fixed, sectioned and stained as previously described6 (link)17 (link) or according to the manufacturer’s protocol (395B-1KT Sigma-Aldrich). Immunostainings were performed as previously described6 (link)59 (link). The following antibodies were used at the indicated dilutions: anti-PCNA (1:10,000, Abcam), anti-Bmpr1a (1:300, Abcam), anti-pSmad1-5-8 Ser463-465 (1:500, Santa Cruz), 488 Alexa Fluor Lectin GSII (1:200, Invitrogen) anti-H+/K+-ATPase (1:2, MBL), anti-GIF (1:75, Abnova), anti-somatostatin (1:100, Santa Cruz), anti-gastrin (1:100, Chemicon), anti-CD3 (1:200, Dako), anti-F4/80 (1:100, eBioscience), anti- α-Sma clone 1A4 (1:5000, Sigma), anti-Vimentin^XP D21H3 (1:100, Cell Signaling), anti-fibronectin (1:1000, Millipore), anti-collagen-I (1:200, Thermo Scientific), anti-collagen-IV (1:200, Chemicon International) and FITC-conjugated anti-rabbit IgG (1:300, Vector), FITC-conjugated anti-goat IgG (1:300, Vector) and Alexa 568-conjugated anti-mouse (1:400, Invitrogen). For immunofluorescence, images were captured on a Leica DMLB2 microscope using a Leica DC300 camera except for α-Sma/pSmad1-5-8 immunostaining and α-Sma/Bmpr1a where a Confocal Zeiss LSM700 along with Zen Blue software was used. For IHC, images were captured on a Nanozoomer Digital slides Scanner (Hamamatsu).