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.