Liberate antibody binding solution

Tissue samples from the mice were fixed with 4% paraformaldehyde for 24 h, decalcified in 12% EDTA for 10 d, embedded in paraffin, sectioned, deparaffinized and stained with hematoxylin and eosin (H&E) and Alcian blue. For IHC staining, antigen retrieval was performed via incubation with Liberate Antibody Binding Solution (Polysciences, USA) for 20 min at RT. Non-specific antigens were blocked using Blocking One (Nacalai Tesque) for 1 h at RT. For antibodies generated from the mouse host, tissues were blocked with ReadyProbes™ Mouse on Mouse IgG Blocking Solution (Invitrogen) for 60 min at RT. Antibodies (diluted in Can Get Signal Immunostain Solution B, TOYOBO) were incubated overnight at 4℃. After rinsing in PBST buffer, samples were incubated with Alexa Fluor secondary antibodies diluted in Can Get Signal Immunostain Solution B for 1 h at RT. A mounting medium with DAPI (Vector Laboratories) was used to counterstain the nuclei. Images were captured using a BZ-X810 microscopy (Keyence, Japan). The antibodies for IHC are listed in Additional file 1: Table S3.

A Human Osteosarcoma Tissue MicroArray (NBP2-30289) was purchased from Novus Biologicals. For antigen retrieval from formalin-fixed paraffin-embedded tissue sections, Target Retrieval Solution, pH 9.0 (S2367, Dako), or Liberate Antibody Binding solution (Polysciences Inc.) was used. Immunoperoxidase staining using the avidin-biotin complex was performed as described previously (64 (link)).

For the detection of acetylated α-tubulin and PIEZO1, cultured cells were fixed with 4% paraformaldehyde at room temperature (RT) for 5 min. For the detection of RUNX2, cells were fixed with acetone at −20 °C for 3 min. For acetylated α-tubulin and RUNX2 detecions, the fixed cells were permeabilized with 0.2% TritonX-100 for 30 min. Then, the blocking was performed with 2% Bovine Serum Albumin (Sigma-Aldrich) in PBS for 1 h at RT for acetylated α-tubulin detection, or with Universal Blocking Reagent (Biogenex) for 6 min at RT for PIEZO1 and RUNX2 detection. The tooth sections were deparaffinized in xylene and rehydrated with water prior to antigen retrieval by Liberate Antibody Binding Solution (L.A.B. Solution, Polyscience) and then washed with PBS. The sections were incubated with Universal Blocking Reagent for 6 min and then with the primary antibodies. The following antibodies were used for immunohistochemistry: mouse anti-alpha Tubulin (acetyl K40) antibody [6-11B-1] (Abcam), rabbit anti-PIEZO1 antibody (Novus Biologicals), mouse anti-DSPP antibody (Santa Cruz Biotechnology, Germany), mouse anti-Runx2 (Cbfa1) mAb (Medical & Biological Laboratories). Alexa Fluor 488- or 594- conjugated secondary antibodies (Invitrogen) were used for detecting the primary antibody. Immunofluorescence was analyzed with an Olympus BX50 microscope (Tokyo, Japan).