Keratinocyte lines stably maintaining HPV16 or HPV18 DNA after electroporation were grown in monolayer culture using E medium in the presence of mitomycin C-treated J2 3T3 feeder cells as described by Meyers and colleagues.18,19,20 Immunofluorescence in L2-Transfected HeLa Cells and Raft Culture Frozen Sections HeLa cells were seeded onto glass coverslips in six-well plates at a density of 1 1 105 per well. expressing high levels of L2, and co-localized with the cellular markers of ND-10, PML, and Daxx. In contrast, L2 was primarily diffuse within the nucleus and distinct from ND-10 as defined by PML immunofluorescent staining in CIN lesions, condylomata, and HPV16-transduced organotypic cultures. Early immunohistochemical studies using polyclonal antisera demonstrate nuclear staining for L2 that is sporadically detected in the upper epithelial layers of condylomata acuminata and cervical intraepithelial neoplasia (CIN).1 In an examination of HPV16+ biopsy specimens, Auvinen and colleagues2 detected L2 protein in 4 of 8 mild dysplasias, 8 of 13 moderate dysplasias, 1 of 1 1 severe dysplasias, but no L2 expression was detected in single cases of carcinoma = 27); squamous cell carcinoma (= 102); adenocarcinoma (= 51); and adenosquamous carcinoma (= 6). Twenty cases of normal cervical tissue specimens were selected from patients undergoing hysterectomy for benign conditions, without a history of CIN or abnormal Pap results. One pathologist (Z.L.) reviewed all of the slides for all those cases YH239-EE independently to confirm the diagnoses. All CIN cases were additionally reviewed by two pathologists (A.V.Y. and B.M.R.) at a multiheaded microscope to establish a consensus diagnosis for these specimens (discrepancies relative to the original diagnoses were resolved by the majority interpretation for each case). HeLa (HPV18-positive), SiHa (HPV16-positive), CaSki (HPV16-positive), ME180 (HPV68-positive), and C33A (HPV-negative) cell lines were obtained from the American Type Culture Collection, Manassas, VA, and cultured in Dulbeccos modified Eagles medium with 10% fetal bovine serum, 100 U/ml penicillin, and streptomycin. Organotypic Raft Culture Primary human foreskin keratinocytes were isolated from neonate circumcision specimens as described previously.19 Keratinocytes were grown in 154 medium (Cascade Biologics, Inc., Portland, OR) supplemented with a human keratinocyte growth supplement kit (Cascade Biologics, Inc.). Keratinocyte lines stably maintaining HPV16 or HPV18 DNA after electroporation were produced in monolayer culture using E medium in the presence of mitomycin C-treated J2 3T3 feeder cells as described by Meyers and colleagues.18,19,20 Immunofluorescence in L2-Transfected HeLa Cells and Raft Culture Frozen Sections HeLa cells were seeded onto glass coverslips in six-well plates at a density of 1 1 105 per well. L2 plasmid was transfected to HeLa cells by using lipofectamine according to the manufacturers recommendations (Invitrogen, Carlsbad, CA). RG-1 (mouse monoclonal generated against HPV16 L217), rabbit polyclonal antibodies PML (1:50, H-238; Santa Cruz Biotechnology, Santa Cruz, CA) and Daxx (1:50, M112; Santa Cruz Biotechnology) were detected using immunofluorescence staining. Cells were fixed with methanol at ?20C for 5 minutes, rehydrated in phosphate-buffered saline (PBS) for 20 minutes, YH239-EE and then blocked in 5% bovine serum albumin-PBST (0.1% Tween-20 in PBS) blocking buffer. Cells were incubated with the primary antibodies diluted in the blocking buffer in the humid chamber for 1 hour at room temperature and then washed three times with PBS. The secondary antibodies of Texas Red-labeled anti-rabbit IgG (H+L) (1:500; Vector Laboratories, Inc., Burlingame, CA) and fluorescein-labeled anti-mouse IgG (H+L) (1:500, Vector Laboratories, Inc.) were incubated for 30 minutes at room temperature followed by three PBS washes. The glass slides were mounted with ProLong Gold antifade YH239-EE reagent with 4,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Eugene, OR). Images were collected using a TE-2000 microscope (Nikon, Tokyo Japan) or an Axiophot confocal microscope (Zeiss, Thornwood, NY). Raft culture frozen sections were fixed in methanol for 5 minutes at ?20C, blocked using 8% bovine serum albumin for 1 hour at room temperature, and then incubated with the primary antibodies for 1 hour at room temperature. The secondary antibodies of Texas Red-labeled anti-rabbit IgG (H+L) (1:500, Vector Laboratories, Inc.) and fluorescein-labeled anti-mouse IgG (H+L) (1:500, Vector Laboratories, Inc.) were incubated for 30 minutes at room temperature followed by three PBS washes, and mounted S5mt with DAPI. Images were collected as above. Immunohistochemistry for L2 in Paraffin-Embedded Tissues and Frozen Sections For an immunohistochemical study with the DAKO LSAB kit (DAKO A/S, Glostrup, Denmark), 4-m-thick tissue sections YH239-EE were deparaffinized, rehydrated, and incubated with 3% H2O2 in methanol for 10 minutes at room temperature to eliminate endogenous peroxidase activity. The antigen was retrieved at 95C for 20 minutes by placing the slides in 0.01 mol/L sodium citrate buffer (pH 6.0). The slides were then blocked by 2% bovine serum albumin followed by incubating.