Anti pnad meca 79

Immunohistochemistry was performed using a modified method of a previously published protocol (78 (link)). Briefly, freshly isolated LNs or spleens were fixed in newly prepared 4% paraformaldehyde overnight at 4°C on an agitation stage. Spleens or LNs were embedded in 4% low melting agarose (Thermo Fisher Scientific) in PBS and sectioned with a vibratome (Leica VT-1000 S) at 30 μm thickness. Thick sections were blocked in PBS containing 10% fetal calf serum, 1 mg/mL anti-Fcγ receptor (BD Biosciences), and 0.1% Triton X-100 (Sigma) for 30min at room temperature. Sections were stained overnight at 4°C on an agitation stage with the following antibodies: anti-PNAd (MECA-79, BD Biosciences) and anti-LYVE-1 (Clone 223322, R&D Systems). Stained thick sections were analyzed using a Leica SP8 confocal microscope equipped with an HC PL APO CS2 40X (NA, 1.30) oil objective (Leica Microsystem, Inc.) and images were processed with Leica LAS AF software (Leica Microsystem, Inc.) and Imaris software 9.3.1 (Bitplane AG).

uLN and dLN were harvested at different dpi, fixed in paraformaldehyde, dehydrated, embedded in OCT-freezing media (Tissue-Tek), cut into 8µ frozen sections, stained with anti-CXCL9 (R&D), anti-PNAD (MECA-79, BD) or anti-ER-TR7 (Santa Cruz). Alexa flour 488 donkey anti-rat and Dyelight 549 Bovine anti-goat (Jackson) were used as secondary antibodies. Images were collected with a Nikon C1 laser Scanning microscope (LSCM) and all the micrographs assembled into a single file using Photoshop software. Contrast and brightness was adjusted in the assembled files to improve visualization in the printed document.