Avidin biotin peroxidase complexes

For histological evaluation, a portion of each UCC patient specimen, xenograft tumor tissue, and mouse lungs were fixed in 10% buffered formalin for paraffin embedding. Paraffin-embedded tissue was sectioned at 5-μm thickness and mounted onto Superfrost Plus^® slides (StatLab Medical Products, Pompano Beach, FL). Sections were used for H&E and IHC staining. For IHC staining, tissue slides were deparaffinized, rehydrated, and blocked for endogenous peroxidase activity. After high-temperature antigen retrieval in 0.01 M sodium citrate solution (pH 6.0), slides were serum-blocked, and immunostained using pre-determined concentrations of primary antibodies (α-SMA, 1 to 300; Ki67, 1 to 200; CD44, 1 to 75; and CD31, 1 to 200) overnight at 4° C. After rinsing the sections in phosphate-buffered saline (PBS), the slides were incubated for 1 hr at room temperature with biotinylated secondary Abs and followed by incubation with avidin-biotin-peroxidase complexes for 1 hr at room temperature according to the manufacturer's instructions (Vector Laboratories). The sections were developed with a 3,3′-diaminobenzidine substrate (Sigma-Aldrich), counterstained with hematoxylin and coverslipped with Permount (Fisher). All images were captured by a deconvoluting microscope using SlideBook 6.0 software (Intelligent Imaging Innovations, Inc, Denver, CO).

CD93 staining was performed using a standard protocol on 4 μm sections from formalin-fixed paraffin-embedded tissue blocks as previously described [24 (link)]. Sections were subsequently incubated with a primary goat anti-human C1qR1/CD93 antibody (0.2 μg/mL; R&D Systems) overnight at 4 °C and then with a horse secondary biotinylated affinity-purified anti-goat IgG antibody (1.5 μg/mL; Vector Laboratories, Ltd., Burlingame, CA, USA). Avidin-biotin peroxidase complexes (Vector laboratories) were added followed by visualization with 3.3′-diaminobenzidine (Vector Laboratories). Sections were counterstained with haematoxylin (Vector laboratories) and rehydrated before coverslips were added. Microscopy of the sections was performed using a Zeiss light microscope (Carl Zeiss Microscopy GmbH, Göttingen, Germany) along with the Zen lite software (Zeiss).