Alexa fluor 594 goat anti rat igg

Animal experiments were conducted in accordance with guidelines set by the Johns Hopkins University Animal Care and Use Committee. Bone and collagen tissue ECM particulate was hydrated with saline (100 mg dry wt/0.2 ml) and injected subcutaneously on the dorsum of 6–8 week old female C57BL/6 mice. Animals were sacrificed after 1 week, implants explanted, fixed in formalin, and embedded in paraffin for sectioning. Sections were then deparaffinized, rehydrated, and immunolabeled for the pan-macrophage marker F4/80 and the M1 marker iNOS. Antigen retrieval was conducted in citrate (10 mM, pH 6) for 30 min in a vegetable steamer, and rinsed with TBS in 0.05% Tween-20. Sections were blocked with 1% bovine serum albumin, 2% goat serum, and 0.05% Tween-20 for 1 hour. Sections were then incubated with primary antibodies against F4/80 (rat monoclonal [BM8] diluted 1:100, ab16911, abcam) and iNOS (mouse monoclonal [4E5] diluted 1:200, ab129372, abcam) overnight at 4°C in blocking solution. Sections were washed and probed with FITC goat anti-rabbit IgG (1:250, Jackson Immuno) and Alexa Fluor-594 goat anti-rat IgG (1:250, Invitrogen) secondary antibodies diluted in blocking solution for 1.5 hours. ECM autofluorescence was then blocked by treatment with 0.1% Sudan Black B in 70% ethanol for 20 min. Sections were counterstained with DAPI for 5 min, washed, coverslipped, and imaged.