Alexa fluor 546 conjugated streptavidin

After dewaxing and hydration, a pressure cooker (2100-Retriever Aptum, Southampton, UK) was used at 120°C for 20 minutes with 10mM of citric acid with a pH of 6.0 for antigen retrieval followed by non-specific binding blocking for 55 minutes, using nonimmune serum matching the secondary antibody. Slides were incubated overnight with CD-31 specific antibody (Abcam, ab28364), anti-cleaved caspase 3 antibody (Cell Signaling Technology 96615) or the appropriate isotype-matched negative control IgG (Vector, I-1000, Burlingame, CA). The primary antibody was localized by a biotinylated secondary antibody (Vector, BA-1000). To localize the antibody complex, Alexa Fluor 546–conjugated streptavidin (Invitrogen S-11225, Carlsbad, CA) was used. Nuclei were counterstained with DAPI (Sigma-Aldrich, St. Louis, MO). Images were captured at different magnifications (40x, 200x, and 400x magnification) using a fluorescence microscope (ECLIPSE 90i; Nikon). The exposure time was set so that the IgG control had no signal. The quantification was performed with the aid of NIS Elements AR image analysis software. The unit of measure was the animal. Each value was calculated by examining six to eight animals in each group.

Spleen and ManLNs were embedded in OCT compound (Sakura Fineteck) and frozen in liquid N₂. Frozen section (5 μm) was fixed with cold acetone, and blocked with PBS containing 5% of normal rat serum and Avidin/Biotin Blocking Kit (Vector laboratories). Subsequently, slide was stained with Alexa Fluor 488-conjugated anti-B220/CD45R mAb, biotin-conjugated PNA (MBL, Nagoya, Japan), followed by Alexa Fluor 546-conjugated Streptavidin (Invitrogen) and Alexa Fluor 647-conjugated anti-CD3ε mAb, and mounted with ProLong Diamond (Thermo Fisher Scientific). The stained slides were analyzed with BZ-X710 fluorescence microscope (KEYENCE, Osaka, Japan).

Paraffin-embedded, formalin-fixed sample sections were processed for immunofluorescence analyses. Antigen retrieval was performed in 10 mmol•L⁻¹ of citric acid, pH 6.0, at 120 °C. Sections were incubated with primary antibody to ß-galactosidase (rabbit, bs-4960R; Bioss Antibodies, Woburn, MA) or RANKL (Goat, sc 7628, Santa Cruz Biotechnology, Inc., Dallas, TX) overnight at 4 °C, as well as the appropriate isotype-matched negative control IgG. Biotinylated secondary antibody (Thermo Fisher Scientific, Waltham, MA) and ABC reagent (Vector Laboratories, Burlingame, CA, USA) were then used. Tyramide signal amplification (Adipogen, San Diego, CA) was also used to enhance the chromogenic signal. Finally, Alexa Fluor 546–conjugated streptavidin (Invitrogen, Carlsbad, CA) and DAPI-containing mounting media (Sigma-Aldrich, St. Louis, MO) were used to visualize the staining. Images were taken at 20× and 40× magnification with a fluorescence microscope (ECLIPSE 90i; Nikon) with the same exposure time for experimental and negative control groups. Image analysis was performed using a NIS Elements-AR software (Nikon). In all experiments capture times were selected so that no immunofluorescence was detected with control IgG.