Rabbit anti tlr7

Dendritic cells were differentiated on 12 mm glass coverslips in 24-well plates, as described before. Infection with the B. abortus was performed at a MOI of 100:1. After 24 h of infection, cells were washed twice with PBS and fixed in 4% paraformaldehyde, pH 7.4, at 37°C for 15 min. Once fixed, cells were incubated with the primary antibody rat anti-lysosomal-associated membrane protein 1 (LAMP-1) diluted 1:200 (Abcam, Cambridge, UK) and rabbit anti-TLR7 diluted 1:100 (Abcam, Cambridge, UK) in PBS 0.1% saponin overnight. Then, cells were washed three times with PBS 0.1% saponin and were incubated for 1.5 h at room temperature with an anti-rabbit secondary antibody conjugated to Alexa 546 (Invitrogen, Carlsbad, CA, USA) and with an anti-rat secondary antibody conjugated to Alexa 488 (Abcam, Cambridge, UK), both diluted 1:1,000. The cells were washed twice with PBS 0.1% saponin and once with PBS then mounted with the mounting medium ProLong Gold with DAPI (Invitrogen, Carlsbad, CA, USA). Stained cells were examined by confocal microscopy (Nikon C2 confocal microscope) and image analysis was performed using ImageJ software version 1.47n.

To measure protein expression levels, spleen and kidney samples were washed with phosphate-buffered saline and then homogenised in Pro-Prep solution (iNtRON Biotechnology). In order to efficiently use the limited samples and avoid the non-specific back ground, membranes were cut prior to hybridization with antibodies. Donkey anti-CTSS (C19, Santa Cruz Biotechnology, CA, USA), rat anti-MHC class II (IBL-5/22, Santa Cruz Biotechnology), rabbit anti-TLR7 (Abcam), rat anti-F4/80 (Cl:A3-1, Bio-Rad, CA, USA), rat anti-Gr1 (RB6-8C5, Bio-Rad) and mouse monoclonal ß-actin (C4, Santa Cruz Biotechnology) antibodies, followed by HRP-conjugated anti-goat IgG (Santa Cruz Biotechnology), anti-mouse IgG (Santa Cruz Biotechnology) and anti-rat IgG (Sigma), were used. Using a Davinch-Chemi™ enhanced chemiluminescence detection system (GE Healthcare, Munich, Germany), immunoreactivity was measured, and the staining intensity was quantified using ImageJ software (National Institute of Health).