Envison flex target retrieval solution

Deparaffinized sections were incubated with antibodies to CD44 (ab157107, Abcam, Cambridge, UK); STRO-1 (ab57834, Abcam), osteocalcin (OC, ab198228, Abcam), osteopontin (OPN, ab218237, Abcam), dentin sialophosphoprotein (DSPP, ab216892, Abcam), alkaline phosphatase (ALP, ab216892, Abcam), pan-cytokeratin (pan-CK, CF190321, TrueMAB™ Thermo Fisher Scientific), vimentin (Vim, MA5-11883, Thermo Fisher Scientific). Antigen demasking was performed before immunohistochemical staining using low pH EnVison FLEX Target Retrieval Solution (Dako, Glostrup, Denmark A/S) at 97 °C for 20 min. Endogenous peroxidase and host IgG were blocked. Primary antibodies were diluted according to the manufacturer’s recommendations. Incubation was carried out for 12 h at 4 °C. An EnVision FLEX detection system (Dako, Glostrup, Denmark A/S) with 2,3-diaminobenzidine DAB chromogen (DAB Chromogen Solution, Dako) was used for imaging. The nuclei were counterstained with hematoxylin. We used incubation without primary antibodies as a negative control.

The cultures of PDLSCs and PSCs were incubated with antibodies to CD73 (MA5-15537, Invitrogen, Waltham, MA, USA); CD90 (ab133350, Abcam, Cambridge, UK); STRO-1 (39-8401, Invitrogen). Deparaffinized sections were incubated with antibodies to osteopontin (OPN, ab218237. Abcam), osteocalcin (OC, ab198228, Abcam), and dentin sialophosphoprotein (DSPP, ab216892, Abcam). Prior to immunohistochemical staining, antigen retrieval was performed using Dako PT Link (Dako, Denmark A/S) at 97 °C for 20 min. The unmasking was performed under low pH using EnVison FLEX Target Retrieval Solution (Dako, Glostrup, Denmark A/S). Endogenous peroxidase and host IgG were blocked, and the primary antibodies (dilution 1:200) incubated for 12 h at 4 °C. The EnVision FLEX Detection System (Dako, Denmark A/S) with the chromogen 2, 3-diamino-benzidine DAB (DAB Chromogen Solution, Dako) was used for imaging. Hematoxylin was used to counterstain nuclei. Incubation without primary antibodies was used as a negative control.