Structural analysis of herpes simplex virus by optical super-resolution imaging

Laine, Romain F.; Albecka, Anna; van de Linde, Sebastian; Rees, Eric J.; Crump, Colin M.; Kaminski, Clemens F.

Antibodies preparation and labelling. Monoclonal antibodies against VP16 (LP1; Abcam (Ab110226)), gD (LP2) and VP1/2 (CB4) were described previously 14, 62, 63 . To generate HSV-1 pUL37-specific monoclonal antibodies, female BALB/c mice were infected with HSV-1 (strain 17) by ear scarification followed by an intraperitoneal boost 1 month later. Spleens were harvested 3 days later, and B-cell hybridomas were generated as previously described 64 . Hybridoma supernatants were screened for reactivity in immunofluorescence assays by using cells transfected with an HSV-1 pUL37 expression plasmid. A cloned hybridoma line secreting antibodies with a strong reactivity to HSV-1 pUL37 was selected and named CB8. Secondary AF antibodies were purchased from Molecular Probes, anti-mouse IgG1 conjugated to ATTO 532 were from Rockland antibodies and assays. AF647 succinimidyl ester was from Molecular Probes. Monoclonal antibodies were purified from hybridoma supernatants using Protein A or G sepharose 4B fast flow from Sigma. Fab fragments were prepared using Pierce Mouse IgG1 Fab and F(ab 0 ) 2 preparation kit for LP1 (IgG1) and Pierce Fab Preparation Kit for LP2 (IgG2a), CB8 (IgG2a) and CB4 (IgG2b), following the manufacturer's instructions. Primary antibodies and Fab fragments were labelled with succinimidyl ester AF probe as follows. Probe was dissolved in dissolved in dimethylformamide to concentration 1 mg ml À 1 immediately prior reaction. Molecular ratio of probe to protein was set to 2.5. One hundred micrograms of antibody/Fab was incubated with probe in 50 mM NaHCO 3 for 1 h. Labelled antibody was purified from unbound dye directly after reaction using NAP5 Sephadex G-25 column from GE Healthcare. Average degree of labelling was between 1.0 and 1.5, as determined using instructions from Molecular Probes.