4 hr post antibody injection, on day 0, mice were vaccinated with 2.5 × 10 irradiated CPS parasites.
Beginning on day 3 post antibody cocktail injection (prior to signs of disease), cohorts of CAIA mice (n = 12 per group) were treated daily (8 or 15 days) with either prednisolone (10 mg/kg per oral, twice daily), a p38 MAPK inhibitor (SD0006; 15 mg/kg per oral, twice daily), and celecoxib (15 mg/kg per oral, twice daily).
41 hours-post antibody treatment, live and dead cells were quantified with the LIVE/DEAD viability/cytotoxicity kit (Molecular Probes), following manufacturer's instructions.
During the past 10 years, the most validated scaffolds have reached the clinical development phase and, recently, one of them [Kalbitor® (Dyax)] has made it to the market, making these alternative scaffold proteins viable drug candidates in a post-antibody landscape.
After incubation, sections were washed and incubated with post-antibody blocking solution (Immunologic, Duiven, The Netherlands) for 15 min (RT).
Embryos were incubated overnight at 4°C with anti-dioxygenin-alkaline phosophatase antibody with rotation before post-antibody wash steps and colour reaction.
The α-SMA antibody (α-SMA/1, M851, 1A4 clone; Dako, Glostrup, Denmark) was applied for 60 minutes followed by post-antibody blocking (Immunologic) for 15 minutes.
After post-antibody blocking, goat poly-horseradish peroxidase (HRP) anti-mouse IgG (Immunologic, Duiven, The Netherlands) was added for 30 minutes.
Importantly, post-antibody therapy tumor samples uniformly exhibit marked downregulation of PVRL4, confirming that the observed response is target-specific and not a consequence of a non-specific antitumor effect of the antibody.
Next morning, sections were washed and incubated for 20 min in post-antibody blocking solution (Immunovision Technologies) before addition of Poly-HRP anti-mouse/rabbit IgG 2° antibody (Immunovision Technologies) for 30 min. Sections were developed in 3,3′-diaminobenzidine (DAB) for 10 min and counterstained with hematoxylin for 1 min before dehydration and mounting.
Optical images were taken at 24 hours post the antibody injection with Xenogen IVIS-200.
