Anti rat igg hrp

Cells were transfected with Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. Briefly, cells were plated at 20%–30% density in 12‐well plates 24 h prior to transfection. For siRNA transfection, the equivalent of 200 nM of siRNA per well of a 12‐well plate was utilized. After a 48 h incubation period, the lysates of the cells were measured using the Bradford assay. Equal amounts of protein were separated by SDS‐PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were immunoblotted with the following specific antibodies: anti‐HSP70, anti‐rabbit IgG‐HRP (Sigma‐Aldrich), anti‐rat IgG‐HRP (Sigma‐Aldrich), and anti‐GAPDH (Sigma‐Aldrich), using standard protocols. The blots were developed with West Dura chemiluminescent substrates using a Bio‐Rad ChemiDoc imaging system.

To assess the pharmacokinetics of L19-mIL12 (1.2 mg/kg) in blood, mice bearing MC-38 tumors were injected intravenously and euthanized at different time points (n = 3/time point). Fresh blood was collected in heparin tubes (BD Microtainer LH tubes) and centrifuged. Plasma was frozen and stored at −80 °C. The concentration of L19-mIL12 was measured by ELISA. 96 well plates (MaxiSorp, Sigma) were coated with EDB (100 nM), followed by incubation with plasma samples for 2 h. L19-mIL12 was detected with anti-mouse IL12 (p70) antibody (1:200, clone C18.2, Biolegend) followed by anti-rat-IgG HRP (1:1000, Sigma). Analysis for each condition was carried out with n = 3.