72 Table 1 Primers used for reverse transcription- polymerase chain reaction Cell Biol Toxicol (2011) 27:69–81 Name and Gene bank accession number Fragment Sequence size (pb) KRT1 NM 006121 KRT10 DQ 896675 KRT13 DQ 890652 KRT15 DQ 896238 KRT19 DQ 895373 HSC70 Y00481 c/EBP alpha NM4364.2 Actin DQ 894128 266 427 440 390 462 200 246 350 P1 Fw TGGATTTGGAAGTCGGAGTC P2 Rv GAGGGAGCAGACGGACCATAA P1 Fw GGCTGCGGTAGGTTTGAATTT P3 Rv GGGTGTTGATCTGACTCAAC P1 Fw CCAGCAGCATCTTGTACTCTTGGT P2 Rv TGAAGGAATTTAGCAACCAGGTGG P3 Fw TGGTTCTGAGCCTCCATCTCGCAT P4 Rv AGGTCAATGTGGAGATGGACGCAG P1 Fw GAGGTGGATTCCGCTCCGGG P2 Rv ATCTTCCTGTCCCTCGAGCAG P1 Fw AGCATCGTTTAAGACACCGT P2 Rv AAGTTTGCAACCCCATCATC P1 Fw AACCTTGTGCCTTGGAAATG P2 Rv CCCTATGTTTCCACCCCTTT P1 Fw AAACTGGAACGGTGAAGGTG P2 Rv TCAAGTTGGGGGACAAAAAG Ta (°C) 60 60 65 69 66 55 59 60 served as a control for absence of genomic DNA contamination. In addition, amplification of beta-actin sequences from the same samples served as positive controls. All PCR reactions were performed with a Perkin Elmer Gene Amp PCR system 2400 (Boston, MA, USA). The PCR products were separated on 2% agarose gel, stained with ethidium bromide, and photographed under ultraviolet illumination. Western blot analysis HaCaT cells were treated with 0.5 and 5 mM NaF during 24, 48, and 72 h. Cells were harvested, and total protein was extracted in 2× denaturing buffer (Prosieve 50 protocol, Cambrex BioScience, Rock- land, ME, USA). The mix was heated for 15 min at 95°C, sonicated, and centrifuged. Bradford technique was used for proteins dosage; equal amounts were electrophoresed on 12% acrylamide sodium dodecyl sulfate (SDS) gels. The separated bands were trans- ferred onto PVDF hybond membranes (Amersham Biosciences, Freiburg, Germany). The first antibody (Table 2) was incubated for 1 h at 24°C. Actin antibody was added to each membrane and served as positive internal control. After washing, the mem- branes were incubated with goat anti-mouse horse- radish phosphate labeled immunoglobulins (DAKO, Glostrup, Denmark) for 1 h at 24°C. After washing, Super Signal West-Pico Luminol/Enhancer solution served as chemiluminescent reagent (Pierce, Rock- ford, IL, USA). The reaction was visualized on Kodak Biomax XAR film. Immunocytochemistry HaCaT cells grown on glass cover slides were treated with low or high dose NaF during 24, 48, and 72 h. Treatment was started when cells had attained 90% confluency. For the reversibility test, cells were kept with normal growth medium and 10% FBS after the fluoride treatment. Immunocytochemistry was per- formed after washing of slides in PBS and fixation in −20°C precooled methanol/ethanol (1:1 v) for 5 min. Slips were incubated with primary mouse monoclonal antibodies for 1 h at 37°C (Table 2). The reaction sites were detected by alexa 594 fluor labeled donkey anti-mouse immunoglobulin G (Molecular Probes, USA). Samples were mounted in Fluoprep aqueous solution (BioMérieux, Marcy l'Etoile, Antigen Antibody clone MW (kDa) Distributor Table 2 Antibodies K1/10 LH1 K13 1C7 K15 LHK15 K19 B170 Beta-actin AC-74 HSP70 A5A 68 (K1); 56.5 (K10) Acris 54 Sigma 52 Novocastra 40 Novocastra 42 Sigma 70 Abcam