Anti rabbit alexafluor 488 labeled secondary antibody

For indirect immunofluorescence staining, shrimp hemocytes were washed twice with anticoagulant and finally exposed in vitro to 0.5 mg/mL of E. coli LPS as previously described [5] . After 1 h the hemocytes were extended on poly-Lysine treated coverslips and then fixed in cold acetone for 10 min. Cells were then washed with PBS, blocked with BSA 3% in PBS for 30 min and incubated with the primary antibody diluted in PBS (1:400 of purified anti-Pa 31 kDa-NOS serum) at 37 C for 75 min. Cells were then washed with PBS for 30 min at room temperature and incubated with anti-rabbit Alexa fluor 488 labeled secondary antibody (Molecular Probes) at 37 C for 45 min. Finally, cells were washed twice in PBS and mounted in PBS-glycerol containing DAPI at 0.1 mg/mL. A Zeiss Axiophot microscope equipped with a 63 Â NA 1.3 oil-immersion objective was routinely used. Images were taken with a SPOT Camera (Diagnostic Instruments Inc.) using the same settings, and processed with Adobe Photoshop CS6.