forward and side scatter profiles. Mature eosinophils were identified as CD45+CCR3+SSChigh; T lymphocytes were iden- tified as CD45+CD3+ cells; CD4+ T cells were identified as CD45+CD3+CD4+ cells. Flow cytometric cell cycle analysis of newly produced and proliferating T helper cell subsets. Lung cells were collected as described above for the staining of different T helper cells, Th1 cells (CD4+CD25+T-bet+), Th2 cells (CD4+CD25+GATA-3+), Th17 cells (CD4+CD25+RORct+) and Treg cells (CD4+ CD25+Foxp3+). Cells were stained with following antibodies; FITC labelled anti-CD4 (clone RM4-5, BD Biosciences) and Alexa FluorH 700 labelled anti-CD25 (clone PC61, BioLegend, San Diego, CA) or a matching isotype control antibody. Cells were fixed and intracellular staining performed according to the manufacturer’s protocol for the Foxp3 staining buffer set (eBioscienceTM). Intracellular antibodies used included PE labelled anti-T-bet (clone eBio4B10, eBioscienceTM), PE labelled anti-GATA-3 (clone TWAJ, eBioscienceTM), PE labelled anti- RORct (clone AFKJS-9, eBioscienceTM) and PE labelled anti- Foxp3 (clone FJK-16s, eBioscienceTM). The subsequent intracellular staining for BrdU and 7-AAD was performed using BrdU Flow Kits (BD PharmingenTM, San Diego, CA) according to the manufacturer’s instructions. BrdU was used to identify the newly produced cells and 7-AAD was used as a DNA dye to identify the proliferation of newly produced cells in situ. All flow cytometry analyses were carried out using a BD FACSAriaTM Flow Cytometer (BD Biosciences, San Jose, CA) with 150 000 events being acquired per sample and analysed with FlowJo SoftwareH (Tri star Inc, Ashland, OR, USA). Due to the cell size and characteristic profile, the non-granulocytes (mononuclear cells) were gated under the intact cells, as described above. CD4+ cells were gated based on forward and side scatter characteristics and isotype control. Lung histology The left lobe of the lung was embedded in Tissue-TekH O.C.T. Compound (Miles, Inc., Elkhart, IN, USA), snap-frozen and stored at 280uC until sliced into 5 mm sections using a cryostat Leica CM1900 UV-cryostatH (Leica Microsystems Nussloch GmbH, Germany). Eosinophils Eosinophils were identified by immunostaining of the major basic protein (MBP), an eosinophil granule protein expressed early in eosinophil-lineage committed cells, as well as in mature eosinophils. (Kind gift from Dr James Lee, Mayo Clinic, Scottsdale, AZ, USA). The immunostaining was performed as described previously [10]. Th1 and Th2 lymphocytes Tissue sections were fixed for 30 min in 2% formaldehyde, rinsed in Tris-buffered saline (TBS) and subjected to heat-induced antigen retrieval using Target Retrieval solution, pH 6 (DakoCy- tomation, Glostrup, Denmark). Endogenous peroxidase was blocked with pre-heated (37uC for 30 min) glucose oxidase solution (PBS supplemented with 0.0064% sodium azide, 0.18% glucose, 0.1% saponin and 1.55 units of glucose oxidase/ml PBS)(all from Sigma) by incubation for 30 min. All washes between the steps were performed using TBS, with or without Tween 20. For T-bet staining, the blocking of unspecific binding sites was performed by incubating the slides with Normal Horse Serum (ImmPRESSTM REAGENT, Vector Laboratories, Inc., Burlin- game, CA). Primary antibodies for the detection of T-bet (rabbit Lung Expansion of Th1, Th2, Th17 and Tregs anti-mouse T-bet, clone H-210, Santa Cruz Biotechnology Inc.) were applied, followed by a secondary antibody-anti-rabbit Ig- (ImmPRESSTM REAGENT, Vector Laboratories, Inc). Bound antibodies were visualised by 3.39-diaminobenzidine (DAB) (DAKO Liquid DAB Substrate-Chromogen System, DakoCyto- mation) and counterstained with Mayer’s Hematoxylin (Sigma). A matched isotype control was used at the same protein concentra- tion as the primary antibody. For GATA-3 staining, the endogenous biotin was blocked by using the Avidin/Biotin Blocking Kit (Vector Laboratories, Inc.). The biotinylation of the primary antibody, GATA-3, (Mouse anti-GATA-3, clone L50–823, BD PharmingenTM) was prepared by the addition of the antibody to the biotin reagent (DakoCytomation ARKTM (Animal Research Kit)) which was incubated for 15 min, followed by the addition of the blocking reagent. Sections were incubated in the prepared biotinylated antibody mixture for 20 min, followed by horse radish peroxidise (HRP) conjugated streptavidin. The detection of the antibody staining and subsequent counterstaining was performed as above for T-bet. T regulatory lymphocytes For Foxp3 staining, the sections were subjected to heat-induced antigen retrieval using Antigen Retrieval Reagent Basic (R&D Systems Inc., Abingdon, UK) for 30 min. The blocking of endogenous peroxidase was performed as described above, while the blocking of unspecific binding sites and endogenous biotin was performed by incubating the slides with 10% Normal Donkey Serum (Jackson ImmunoResearch Laboratiories. Inc., PA, USA) followed by the use of the Avidin/Biotin Blocking Kit. The primary antibody for the detection of Foxp3 (rat anti-mouse/rat FOXP3, clone FJK-16s, eBiosciencesTM) was applied, followed by the secondary antibody, biotin-conjugated F(ab’)2 donkey anti-rat IgG (Jackson ImmunoResearch Laboratories. Inc.) and ExtrA- vidin-HRP (Sigma). Antibody detection and counterstaining was performed as above. Th17 lymphocytes For RORct staining, tissue sections were fixed with 2% formaldehyde for 10 min, rinsed in PBS and incubated with 10% Normal Rabbit Serum (Jackson ImmunoResearch Labo- ratories, Inc.), followed by incubation with rat anti-mouse/ human RORc (t) (ROR gamma, Retinoid-Related Orphan Receptor gamma) (clone AFKJS-9, eBioscienceTM). Unless otherwise stated, a TBS-saponin solution was used for all wash steps. The reaction was visualised by using the APAAP (alkaline phosphatase anti-alkaline phosphatase) technique with the bridge antibody, rabbit anti-rat IgG (DAKO, Z0494) and monoclonal APAAP rat IgG (DAKO, D0488), with each antibody being applied for 30 min. To increase the staining intensity, these two steps were repeated. After a final wash with TBS, bound antibodies were visualised using Liquid Permanent Red Chromogen (LPR) (DakoCytomation). Counterstaining was performed as above. Quantification The stained samples were assessed in a blinded fashion using an Axioplan 2 microscope (Carl Zeiss Jena GmbH) at a magnification of x400. To facilitate counting, a graticule was applied to the ocular. Eight representative sections of each of the three lung microenvironments (peribronchial, perivascular and alveolar tissue), were assessed. Positive cells were counted and the data expressed as the number of cells/mm2 [11]. PLoS ONE | www.plosone.org 3 May 2011 | Volume 6 | Issue 5 | e19889