Detection of BsAbs in cynomolgus monkey liver
Liver biopsy samples from the left lobe of each animal were collected from the aforementioned biodistribution study for immunofluorescence analyses to determine the cellular disposition of BsAb-1 and BsAb-2 at 1 hour post administration using previously reported methods.13 Stored formalin fixed and paraffin embedded (FFPE) liver tissue from previously reported experiments of a ECD-based BsAb were aslo included as a positive control for sample processing and the detection of molecule in tissue.13 Briefly, liver tissues were processed to FFPE and sectioned as previously described.13 FFPE sections were deparaffinized and rehydrated prior to immunofluorescent staining to BsAb-1 or BsAb-2, as well as, endothelial cells (CD31 marker).32, 33Antigen retrieval was performed using 1x heat-induced epitope retrieval solution, Diva Decloaker (BioCare Medical, Concord, CA) for 30 seconds at 125oC under pressure. After rinsing with 1x TBS-T (Dako Wash Buffer, Dako Carpinteria, CA), liver sections were incubated with a polyclonal anti-human IgG (Bethyl Laboratories, Montgomery, TX, A80-319A) at 10 µg/mL to detect antibodies and a monoclonal anti-human CD31 (CD31/PECAM1 R&D Systems AF806) at 10 µg/mL. Species-specific control IgG obtained from Jackson ImmunoResearch, (West Grove, PA) or R&D Systems, (Minneapolis, MN), respectively, were used at concentrations equivalent to the primary antibodies as a control to determine the specificity of BsAb detection.  Following incubation with the primary antibody or control IgGs, slides were rinsed and followed by detection with donkey anti-species Alexa dye conjugated reagents each at 10 µg/mL (Life Technologies, (Grand Island, NY) or Jackson ImmunoResearch (West Grove, PA). Stained slides were coverslipped using Dako fluorescent mounting media.  Reagent volumes ranged between 150-200 uL/slide, dependent upon tissue area to be covered.  All steps were performed at ambient temperature having slides and reagents protected from light.
Images of the stained slides were collected on a 3-D HISTECH (Budapest, Hungary) scanner having Plan-Aplchromat 40x objective lenses at ambient temperature.  The fluorochromes used were Alexa-488 and Alexa-555. A PCO.edge camera was used to capture images in the JPEG medium and the Pannoramic Viewer software (3DHISTECH) was used for image acquisition. Brightness and contrast parameters were applied consistently to all images.