Metamorph 7 imaging software

Tumor pieces were fixed overnight with Periodate-Lysine-Paraformaldehyde [48 (link)] at 4°C and Immunofluorescence on tumor slices was performed as previously described [17 (link), 49 (link)]. Immunostaining was performed by first blocking Fc receptors with anti-FCR (BD Pharmingen), then staining for 1 h RT or at 4°C overnight was performed with primary antibodies specific for CD8-PerCP, CD45-APC, IA-IE-PE, CD11b-FITC, Ly6C-APC, CD31-biotin (all from BD Pharmingen), fibronectin, gp38/podoplanin, F4/80-biotin (all from Biolegend) or F4/80-PE (AbD Serotec). Immunodetection was performed using anti-Rat or anti-rabbit antibodies coupled to 488, 568 or 647 (BD Pharmingen) and streptavidin- Alexa Fluor 488 or 647 (Invitrogen). Slices were then counter-stained with Hoechst for 10 min at room temperature. Antibodies were diluted in PBS, 0.5% BSA, 2% human serum. Images were obtained with a spinning disk microscope equipped with a CoolSnap HQ2 camera (Photometrics) and a 20x and a 63x objective. All images were acquired with MetaMorph 7 imaging software (Molecular Devices) and analysed with ImageJ. For staining of dying cells in vivo, vaccinated TC1-GFP bearing mice received an intra-tumoral injection of propidium iodide (1 mg/ml) and were sacrificed 30 min later.

Spleens from 14 day-immunized mice were initially fixed with paraformaldehyde and embedded in 4% low-gelling-temperature agarose (type VII-A; Sigma-Aldrich) prepared in PBS. 150-μm slices were cut with a vibratome (VT 1000S; Leica) in a bath of ice-cold PBS. For immunolabeling, samples were saturated with PBS supplemented with 10% of fetal calf serum, then were labeled with primary antibodies anti-B220-APC (clone RA3-6B2) and anti-IgD-PE (clone 11-26c.2a) and analyzed with a spinning disk confocal microscope equipped with a CoolSnap HQ2 camera (Photometrics) and a 20x objective. Images were acquired and analyzed with MetaMorph 7 imaging software Molecular Devices).