Rabbit anti klf10

Cells were fixed in cold acetone and methanol (1:1), permeabilized in 0.2 % Triton X-100 at ice for 30 min and blocked with 10 % goat serum for 30 min at RT. For double-labeling, two primary antibodies were incubated simultaneously overnight at 4 °C at the following dilutions: rabbit anti-Klf10 (1:100; Sigma-Aldrich), goat anti-Dsp (1:100; Santa Cruz Biotechnology), goat anti-Runx2 (1:100; Santa Cruz Biotechnology) and mouse anti-Dmp1 (1:100; kind gift from Dr. Chunlin Qin, Texas A&M University, Baylor College of Dentistry, Tex., USA; Qin et al. 2006 (link)). After being washed, slides were incubated with the secondary antibody conjugated with Alexa Fluo 486 green and Alexa Fluo 568 red (1:500; Molecular Probes) for 1 h at RT. As a negative control, the primary Klf10 antibody was replaced by mouse IgG I. For nuclear staining, the cells were treated with Hoechst (Sigma-Aldrich). Slides were viewed and captured by using a Nikon inverted microscope. For double-fluorescent immunohistochemistry, the co-expression of Klf10 and PCNA (proliferating cell nuclear antigen) was analyzed in tissue sections from various mouse ages. Anti-PCNA antibody was purchased from Santa Cruz Biotechnology.

iMDP-3 cells induced for various times (0, 1, 3, 5, 7, 11, and 14 days) were lysed with RIPA buffer (Santa Cruz Biotechnology) at 4 °C by vigorous shaking for 15 min. After centrifugation at 12,000g for 15 min, the supernatant was separated and stored at −80 °C until use. The protein concentration was measured by using a Bio-Rad protein assay kit (Bio-Rad Laboratory). An equal amount of proteins was loaded onto a 10 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE) system and the bands were subsequently transferred to a membrane (Bio-Rad Laboratory). Rabbit anti-Klf10 (Sigma-Aldrich) and goat anti-actin (Santa Cruz Biotechnology) were used as primary antibodies. Transferred proteins were incubated with ECL substrate solution (Thermo Scientific) according to the manufacturer’s instructions and labeled bands were revealed on X-ray film.