Hrp conjugated streptavidin

For endpoint titers, serum gp120-specific antibody responses were evaluated by enzyme-linked immunoabsorbant assay (ELISA) as described previously [45 (link)] Briefly, EIA/RIA microtiter plates (Costar, Corning, NY) were pre-coated with 5 μg/well ConA for 1 h. After washing (AquaMax2000, Molecular devices, Sunnyvale, CA), plates were coated with 1 μg/ml gp120 protein mix. After blocking overnight (4% whey and 5% powdered milk), plates were incubated with mouse sera for 1 h. For detection of IgG, biotinylated anti-mouse IgG was added followed by HRP-conjugated streptavidin (Vector Laboratories, Burlingame, CA). Serum titers were determined as the highest dilution of immune serum producing ELISA values (A_{450 nm}) greater than or equal to two times the binding detected with a corresponding dilution of pre-immune serum. Plates were coated with 0.5 μg/ml gp120 (Advanced BioScience Laboratories, Kensington, MD). Dilution series of sera was performed. The end titration was determined as the highest dilution at which the absorbance at OD450 equaled twice the absorbance of negative control wells. For isotyping of serum, standard curves for each isotype were performed. The end titration was determined as the highest dilution at which the absorbance at OD450 equaled twice the absorbance of negative control wells.

After dissection whole eyes were embedded in paraffin. Each tissue block was then sectioned at a thickness of 6 μm. Every section was examined with a light microscope until it contained either capsule or regenerated lens, which was then mounted on a glass slide as the “first” section. Then every fifth section was slide-mounted until tissue sections no longer contained capsule or regenerated lens (the “last” section). All slides were then processed for 3-dimensional analysis (described below). For confirmation of fiber cell characteristics, sections from the approximate middle of each eye (ARKO, ARTg, WT, WT with sorbinil) were stained for γ-crystallin. Sections were first processed by antigen retrieval and blocking of endogenous peroxidase activity. Sections were then incubated with a 1:200 dilution γ-crystallin in OP Quanto Diluent (Thermo Fisher Scientific, Waltham, MA, USA) in phosphate buffered saline (PBS) for 1 hour. After three washes with PBS, the sections were incubated with biotinylated secondary antibody (Vector, Burlingame, CA, USA) for 1 hour, then HRP conjugated streptavidin (Vector, Burlingame, CA, USA) for 30 minutes and DAB (Vector, Burlingame, CA, USA) for 10 minutes. Images were acquired immediately after processing.