When the passaged cells reached 80% confluence they were used for growing raft cultures. Raft cultures were grown as previously described [19, 20]. Briefly, mouse fibroblast 3T3 J2 cells were trypsinized and re-suspended to a concentration of 2.5 × 105 cells/mL in 1% reconstitution buffer, 10% 10× DMEM (Life Technologies, Gaithersburg, MD), 2.4 μL of 10 M NaOH and 80% collagen (Dickinson, Franklin Lakes, NJ) on ice. The mixture was then aliquoted into six-well plates, each well containing 2.5 mL of the mixture. The plates were incubated at 37°C for

NVP-BGJ398 2 h to allow the collagen matrices to solidify. Two millilitres of E-medium was then added to each well so that the matrix could equilibrate. Human gingival epithelial keratinocytes were trypsinized and re-suspended in E-medium plus 5 ng/mL epidermal growth factor (EGF) at a concentration of 1 × 106 cells/mL and 1 mL of the suspension was added to the top of each collagen matrix. Epithelial cells were allowed to attach to the collagen for 2–4 h before the medium was removed and the matrices

were lifted onto stainless steel grids. The grids rested at the air–liquid interface and the raft cultures were fed by diffusion from below with E-medium supplemented with ZDV. ZDV capsules (Aurobindo Pharma, Cranberry, NJ, USA) were purchased from the pharmacy at The Milton S. Hershey Selleck 3-deazaneplanocin A Medical Center, Penn State University. The contents of either a 100-mg or 300-mg capsule were removed and re-suspended in sterile PBS. Serial dilutions were made directly in E-medium to obtain the correct concentration. The maximum

level of ZDV reached in the blood of patients, or Cmax, is 2 μg/mL [21-23]. Two additional concentrations on either Exoribonuclease side of the Cmax were also used. In the first set of experiments, the rafts were treated with ZDV at concentrations of 0.5, 1, 2, 4 and 6 μg/mL from day 0. Control rafts were fed with E-medium only. In the second set of experiments the same concentrations of ZDV were used but the rafts were treated on day 8. All rafts were fed every other day and harvested at the time-points indicated in Figure 1. Raft cultures were fixed in 10% buffered formalin, and embedded in paraffin. Four-micrometre sections were cut and stained with haematoxylin and eosin as described previously [19]. Immunostaining was performed with the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) [19]. Briefly, a vacuum oven was used to bake slides at 55°C for 1 h. Tissue sections were then dehydrated in xylene and rehydrated in alcohol gradients. Endogenous peroxide activity was neutralized by incubating the slides in a 3% H2O2 solution. Tissue sections were blocked for 1 h with 3% normal horse serum and 20% normal goat serum for primary mouse antibodies and rabbit antibodies, respectively. Next, slides were incubated in primary antibodies for 1 h.