F4 80 pe

A suspension of 0.5 × 10⁶ cells was washed with cold PBS (phosphate buffered saline, pH 7.2, Gibco) and stained for 30 min at 4 °C with antibodies against surface markers coupled with flurochromes: CD11b-PercP or CD11b-APC, F4/80-PE, MHC class II-FITC, CD80-PE, CD86-APC, Dectin1-PercP Cy5, Mannose receptor (MR, CD206)-FITC, CD23-PE (AbD Serotec, Kidlington, UK) or IL-4Receptor-PE (BD Sciences, San Diego, CA, USA,). Unbound antibodies were washed out, cells were resuspended in 300 μL of PBS and analysed in a FACS Calibur cytometer with CellQuest and FCS Express 4 Flow (BD Sciences, San Jose, CA, USA) programs. Cells were gated into different populations based on F4/80 and CD11b expression. The level of cellular markers was presented as a percentage of positive cells in the CD11b⁺ population. The peritoneal suspension was smeared on a glass slide and stained with Giemsa for cell recognition.

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.