successively in distilled water, 30% isopropanol, and 60% isopropanol. Slides were then incubated in Oil Red O for 15 minutes and subsequently washed in 60% isopropanol, 30% isopropanol, and water. Slides were counterstained with hema- toxylin, rinsed in water for 10 minutes and coverslipped with glycerin jelly. Co-immunofluorescence staining Tissue slides were deparaffinized and hydrated in successive washes (Xylene, 95% ethanol, 70% ethanol, PBS), blocked (K5,K10: 1 h with 1.5% natural goat serum (NGS), 1.5% natural horse serum (NHS), 1% bovine serum albumin (BSA) in PBS. C/EBPa,C/EBPb: 2 h with 6%NGS, 6%NHS, 1%TritonX in PBS), and incubated overnight at 4uC with primary antibodies: K5 (rabbit polyclonal, Covance, 1:1000), K10 (mouse monoclonal, Covance, 1:1000), C/ EBPa (rabbit polyclonal, Santa-Cruz SC-61, 1:400), and C/EBPb (mouse monoclonal, Santa-Cruz SC-7962, 1:200). Slides were washed in PBS and incubated 45 minutes with fluorophore-linked secondary antibodies (Alexa FluorH 568 goat anti-mouse (1:1000), Alexa FluorH 488 goat anti-rabbit (1:1000)) in PBS, dehydrated and coverslipped with Dako Faramount Aqueous Mounting Medium. RNA and quantitative PCR In brief, mice were killed by cervical dislocation and skin removed. Whole skin was minced in Sigma Tri-ReagentH, T- 2494, sonicated on ice 4610 s, centrifuged 14,000 g/10 m/4uC, and RNA was extracted with chloroform. Pelleted RNA was solubilized in water and then purified with DNase on Qiagen RNeasyH columns. cDNA was created with equal amounts of Roles of C/EBPs in Adult Skin starting template with reverse transcriptase with appropriate no- RT controls. cDNA was then subjected to Real Time TaqManH PCR (Applied Biosystems: keratin 10 (Mm03009921_m1), SCD3 (Mm00470480_m1), MC5R (Mm00442970_m1)) using GAPDH (Mm99999915_g1) as a housekeeping gene) and semi-quantitative PCR (all others) using the primers given in Table S1. For semi- quantitative PCR, PCR product was then run on 2% agarose gels with .01% ethidium bromide. After confirming PCR amplification was in the linear range by varying the amount of starting template, optical density of bands were determined by ImageJTM. Each experiment included 3 or more animals of each genotype. Results C/EBPa and C/EBPb are co-expressed in skin The bZIP transcription factors, C/EBPa and C/EBPb are expressed in mouse epidermis and sebaceous glands [23,24,34]. To determine whether C/EBPa and C/EBPb are co-expressed within the same cell and not just within the same tissue or gland we performed co-immunofluorescence labeling for C/EBPa and C/ EBPb in untreated animals. As shown in Fig. 1A, C/EBPa and C/ EBPb are co-expressed in the nuclei of many interfollicular epidermal (IFE) keratinocytes. Most IFE suprabasal and basal keratinocytes that expressed C/EBPb also expressed C/EBPa. However, some basal keratinocytes predominately expressed C/ EBPa. The majority of outer root sheath (ORS) follicular keratinocytes co-expressed C/EBPa and C/EBPb in their nuclei as did the vast majority of sebocytes of the sebaceous gland which displayed the highest levels of staining intensity for C/EBPa and C/EBPb (Fig. 1B). Figure 1. C/EBPa/b are co-expressed in skin and acute ablation C/EBPb does not produce a major skin phenotype. Co-IF staining of untreated mouse skin in: (A) interfollicular epidermis (IFE), and (B) the hair follicle outer root sheath (HF-ORS) and sebaceous gland (SG). (C) Immunoblot analysis of epidermal lysates from untreated and 4OHT-treated C/EBPbff and IKOb mice (n = 3 mice/genotype/treatment). (D) IHC staining for C/EBPb in 4OHT-treated C/EBPbff and 4OHT-treated IKOb mouse skin. (E) H&E staining of 4OHT-treated C/EBPbff and 4OHT-treated IKOb mouse skin. (F) Quantification of nucleated cell layers in 4OHT-treated C/EBPbff and IKOb mice (n = 3 mice/genotype/treatment). (G) Quantification of basal BrdU positive cells in 4OHT-treated C/EBPbff and IKOb mice (n = 3 mice/genotype/treatment). (H) IHC staining for BrdU and markers of squamous differentiation. All scale bars represent 30 microns. *indicates significantly different from controls p,.05, Student’s t test. doi:10.1371/journal.pone.0009837.g001 PLoS ONE | www.plosone.org 3 March 2010 | Volume 5 | Issue 3 | e9837