Goat anti mouse igg1 apc

Suspended cells were incubated with fluorochrome-conjugated monoclonal antibodies (mAbs) on ice for 30 min in the dark. PBS with 2% FBS was used as a washing solution and antibody diluant. A two-step incubation was performed with biotinylated primary antibodies and a streptavidin-labeled fluorochrome reaction. All labeled mAbs and labeled fluorochromes were purchased from Becton Dickinson unless otherwise indicated. The following mAbs and fluorochromes were used as primary antibodies: fluorescein isothiocyanate (FITC)-conjugated anti-human CD66 (hCD66FITC), hCD13FITC, hCD24FITC, hCD29FITC, and hEpCAMFITC (Miltenyi Biotec); allophycocyanin (APC)-conjugated hCD45 (hCD45APC), hCD90APC, hCD81APC, hCD117APC, hLGR5APC (Miltenyi Biotec), and hCD133/2APC (Miltenyi Biotec); phycoerythrin (PE)-conjugated hCDCP1 (hCDCP1PE), hCD34PE, hCD44PE, hCD49fPE, hCD140aPE, hCD166PE, and hCD138PE (Miltenyi Biotec); biotinylated hCD54 (hCD54Bio; eBioscience), hCD55Bio, and hCD56Bio; hDLK (IgG1, Abcam); and rabbit anti-LDLR (Abcam). Secondary antibodies were as follows: streptavidin-labeled APC, goat anti-mouse IgG1-APC, and goat anti-rabbit IgG-Alexa 488 (Invitrogen, Carlsbad, CA, USA). Cell profiling analyses and sorting were performed on a high-speed cell sorter, MoFlo (DakoCytomation).

The infected PCLS were fixed 24 h at 4°C with 4% formalin and then transferred to a 48-well culture plate in PBS. All steps that will be described below were done under gentle agitation at room temperature (RT). The PCLS were incubated for 2 h with 100 μl of PBS, 0.25% Triton X-100, and 10% horse serum for permeabilization and saturation (saturation buffer). They were incubated overnight at 4°C with primary Ab (anti-bovine MHCII clone MCA5655 from BioRad and anti-bovine pancytokeratine clone BM4068 from Acris) diluted in saturation buffer. The PCLS were washed four times with 300 μl of PBS (two times for 5 min and then two times for 10 min) and then incubated for 3 h with fluorescent-conjugated secondary antibodies diluted in saturation buffer (goat anti-mouse IgG1-APC and goat anti-mouse IgG2a A555 from Invitrogen). The PCLS were washed four times with 300 μl of PBS (two times for 5 min and then two times for 10 min), transferred on cover slides which were mounted with Fluoromount-G™ mounting medium containing DAPI (Invitrogen), and sealed with a transparent nail polish. Z-stack imaging was performed at ×63 enlargement with a confocal microscope (LEICA) and analyzed with LAS software. The presence/absence of Mb and number of macrophages per alveoli were numerated by eye at the confocal microscope, with one person counting and the other confirming and reporting the data.