Int. J. Environ. Res. Public Health 2012, 9 476 USA). As3+ stock solutions were freshly prepared for each experiment. A rabbit anti-tubulin antibody and an FITC conjugated anti-tubulin antibody were from Polysciences (Warrington, PA, USA). A mouse anti-metallothionein antibody (Dako-MT, E9) against both metallothionein-I and metallothionein-II were from Dako Inc. (Fort Collins, CO, USA). A rabbit antibody against the heavy chain of -glutamylcysteine synthetase (-GCS) was from NeoMarkers (Fremont, CA, USA). An FITC-conjugated goat-anti-rabbit IgG was from Organon-Teknika (Malvern, PA, USA). A rabbit anti-actin (I-19) antibody and a horseradish peroxidase (HRP)-conjugated goat anti rabbit or mouse IgG were from Santa Cruz Biotech. (Santa Cruz, CA, USA). [3H]NEM (30–60 Ci/mmol) and [35S]methionine (1,000 Ci/mmol) were purchased from PerkinElmer Life and Analytical Sciences, Inc. (Waltham, MA, USA). Oligonucleotide primers for the PCR were synthesized by Integrated DNA Technologies (Coralville, IA, USA). Protein assay reagents for the Bradford dye binding method were from Bio-Rad (Richmond, CA, USA). All tissue culture products were from Invitrogen Co. (Carlsbad, CA, USA). 2.2. Cell Cultures The fibroblastic RFL6 cell line derived from the fetal lung tissue of normal Sprague-Dawley rats was obtained from ATCC. Cells were regularly maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). To standardize experimental conditions, cells were growth-arrested and synchronized at the G1 phase by incubation in 0.3% FBS/DMEM for 72 h followed by changing to fresh 0.3% FBS/DMEM and incubation for another 24 h before experiments [21]. Cells were treated for 24 h with As3+ or other reagents at various concentrations, then processed for further analysis. 2.3. Fluorescence Microscopy MTs were revealed fluorescently in control and treated cells with the primary rabbit anti-tubulin antibody and the secondary goat-anti-rabbit IgG coupled with FITC as described [22]. To assess As3+ effects on cell division, cells on coverslips were incubated in 10% FBS medium without or with 10 µM As3+ for 24 h. After fixation with 3.7% formaldehyde/0.2% Triton X-100/PBS and washing with PBS, cells were treated with an RNase A/PBS solution (the final concentration = 50 µg/mL) to lyse cellular RNA. Cell division stages and aberrations were visualized by the in situ chromosomes staining with propidium iodine (the final concentration = 50 µg/mL in PBS containing 2 mM MgCl2) and spindle MT staining with FITC-conjugated anti-tubulin antibody under the dark condition. Samples were examined under the Nikon fluorescence microscope using the DAPI-FITC-TRITC filter to detect green and red fluorescence simultaneously. All photographs were taken at the same magnification with a 40 × Planapochromat objective. 2.4. Immunohistochemistry and Total RNA Extraction in Lung Tissues of the Rat Animal Model To assess As3+ injury to the lung MTs, eight Sprague-Dawley rats (body weight  150 g) per group were intratracheally instilled with 520–530 µg NaAsO2 in 100 µL physiological saline according to 2.02 mg As/kg body weight once a week for 5 weeks. Control rats received saline only. Rats were