Fitc conjugated goat anti rabbit igg

Paraffin-embedded tissue sections were deparaffinized and rehydrated. Heat-mediated antigen retrieval was performed using Tris/EDTA buffer at a pH of 8. Sections were blocked in PBS with 1% BSA and 10% FCS for one hour and then incubated with rabbit-anti-mouse MTP1/FPN IgG (dilution 1:50; Alpha Diagnostics, Cologne, Germany) at 4°C in a humid chamber overnight. After three washing steps, sections were incubated with FITC-conjugated goat-anti-rabbit IgG (dilution 1:1000; Sigma-Aldrich, Hamburg, Germany). Autofluorescence was reduced with 10% (w/v) Soudan Black B and sections were mounted with Immu-Mount (Thermo Fisher Scientific, Darmstadt, Germany). Slides were examined using an Olympus BX50 microscope and results were documented with Cell^F software (Olympus, Hamburg, Germany).

BM- and AD-ECM were rehydrated by incubation in PBS for 1 h at 37 °C and then treated with DNase (100 units/mL; Sigma-Aldrich) for 1 h at 37 °C. After removal of the enzyme and a brief rinse in PBS, the ECMs were fixed with 4% paraformaldehyde/PBS (Sigma-Aldrich) for 30 min at room temperature. After fixation, the ECMs were washed three times with PBS and then stored in PBS until commencing the staining procedure.
For IF staining, ECMs were first treated with blocking solution (Bloxall, Vector Labs, Inc., Burlingame, CA) for 30 min at room temperature and then incubated for 1 h at 4 °C with primary antibody specific for human type VI collagen (1:200 dilution; polyclonal rabbit IgG; EMD Millipore, Temecula, CA). Non-specific rabbit IgG (Sigma-Aldrich) was employed as a negative control (1:200). Unbound primary antibody was removed by washing with cold (4 °C) PBS. After washing, the ECMs were incubated with blocking solution again for 10 min at room temperature. Secondary antibody (1:200; FITC-conjugated goat anti-rabbit IgG, Sigma-Aldrich) was then added and the incubation continued for 1 h at 4 °C. Florescence microscopy was performed at 10 × magnification using an Olympus IX73 inverted microscope.