Alexa fluor 488 antibody labelling kit

Pristane was injected i.p. in WT and CRAMP^−/− mice and cells were collected via peritoneal lavage with PBS or after erythrocyte lysis from peripheral blood at day 0 and 7 after injection. Cells were stained extracellularly with anti-CD11b-eFluor450 (eBioscience), anti-Ly6C-APC (BioLegend), anti-CD115-PE (BioLegend), and anti-Ly6G-APC (BioLegend) to determine cell types. Intracellular CRAMP staining was performed with an anti-CRAMP (Innovagen) antibody labelled with an Alexa Fluor 488 antibody labelling kit (LifeTechnologies). Cells were then analyzed by cytofluorometry on a Gallios machine (Beckman Coulter).

For surface immunofluorescence cells were seeded as for immunofluorescence and on the day of experiment incubated with primary antibodies in full media at 4 °C for 1 h. Next, cells were washed 3 times in PBS, fixed in solution containing 4% PFA and 4% sucrose in PBS for 8 min, and washed again 3 × 5 min in PBS. Then, cells were blocked in 2.5% BSA in PBS for 1 h, at RT and incubated for a further 1 h with secondary antibodies The remainder of the procedure was the same as described in the “Immunofluorescence” section. Anti-sortilin antibody labelling with Alexa Fluor 488 fluorophore was performed using Alexa Fluor 488 antibody labelling kit (A20181, Life Technologies) according to manufacturers’ instructions.