Tooth pulp cryosections were stained with Fibronectin and FnBPA5 probe as previously described.39 (link) Briefly, thawed sections were washed with ice-cold 1xDPBS and blocked with 0.3 mol·L-1 glycine animal-free blocking buffer (Animal-Free Blocker® and Diluent, R.T.U. SP-5035-100) at room temperature for 30 min. Sections were then incubated with 1 μg·mL-1 Cy5-FnBPA5 or Cy5-labelled scrambled-FnBPA5 (scrambled-FnBPA5) for 1 h, followed by three 5 min washes with Dulbecco’s phosphate-buffered saline (DPBS), second blocking with the blocking buffer and incubation with polyclonal rabbit anti-Fibronectin (ab23750, Abcam) and monoclonal rat anti-Collagen I (7025, Chondrex) antibodies, overnight at 4 °C. The following day samples were extensively washed with DPBS before 1 h incubation with secondary antibodies and 2 μg/ml DAPI at room temperature. The secondary antibodies used were goat anti-rabbit (AF488, A11034, Invitrogen) and goat anti-rat (AF546, A11081, Invitrogen) antibodies. Following extensive washes after incubation with the secondary antibody, the samples were mounted using DAKO Fluorescence mounting medium (DAKO, Denmark) and imaged.
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