C57BL/6 mice were purchased from the Animal Production Area, NCI-FCRDC (Frederick, MD). Rag2−/− were originally purchased from The Jackson Laboratory (Bar Harbor, MN, USA) and maintained by homozygous breeding at NCI-Frederick, MD. Three strains of IL-7-promoter-ECFP (IL7promECFP) mice have been produced at NCI, Frederick MD and homozygous strains have been selected and maintained at NCI-Frederick, MD.
Transgenic mice expressing an IL-7 cDNA under the control of the human keratin 14 (K14) 
promoter were maintained in specific pathogen free (SPF) conditions at the animal facility of the Department of Dermatology, Brigham and Women's Hospital, Boston, MA. Tissues from these animals were removed and processed for histopathology or RNA extraction promptly after euthanasia and shipped overnight on dry packs or dry ice to our facilities.
mice were purchased from Taconic Farms (Germantown, NY, USA) and maintained by homozygous breeding at NCI-Frederick, MD.
NCI-Frederick is accredited by AAALAC International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the Guide for Care and Use of Laboratory Animals (National Research Council; 1996; National Academy Press; Washington, D.C.).
Il7 in situ hybridization
In situ hybridization (ISH) assays were developed that were specific for murine Il7 and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and used to define temporal and special expression patterns in optimally processed adult mouse thymus and lymph node tissue. Sequence verified riboprobe generation templates corresponding to bps 884–1238 of Gapdh (Accession # NM_001001303) and bps 294–705, 792–1185 and 294–1185 of Il7 (Acc # NM_008371) were produced via a PCR based strategy that utilized forward primers flanked by the T3 promoter and reverse primers flanked by the T7 promoter. Corresponding riboprobe pairs (sense and antisense) were then synthesized (Megascript high yield in vitro transcription kit, Ambion), purified (MEGAClear purification kit, Ambion), resuspended at 100 ng/µl in RNA storage solution (Ambion) and held at −80°C until use. Probe size and integrity have subsequently been confirmed via Agilent Bioanalyzer 2100 NanoChip analyses (data not shown). Due to anticipated very low level expression of Il7 mRNA, HEK293 human cell line transfected with a high-level Il7 mRNA expression construct (pECFP-N1/IL-7 or pEYFP-N1/IL-7) were prepared. Untransfected cells, transfected cells and mouse thymi and lymph nodes were harvested and fixed for 24 hrs at 4°C in freshly prepared 4% paraformaldehyde (PFA) in PBS then processed into paraffin blocks. Additional fixed tissue was cryoprotected in 20% sucrose at 4°C for 24 hrs and then embedded in OCT to generate frozen tissue blocks. Ten micron paraffin or frozen sections were then placed onto Super-Frost Plus glass microscope slides (Fisher Scientific) and held at −20°C until ISH. Specificity of each probe was verified in a series of pilot ISH (method described in detail below) on paraffin sections of the transfected and untransfected HEK293 cells (data not shown). Within this, previous work had indicated that Gapdh mRNA is readily detected in HEK293 cells by chromogenic in situ hybridization (unpublished observation). Detection of the corresponding mRNA by ISH is therefore a useful positive control that indicates successful tissue fixation, pre-treatment and in situ hybridizations.
Immediately prior to ISH, representative sections were removed from −20°C, dried at 60°C for 1 hr, de-paraffinized and re-hydrated through graded ethanols into 1X Phosphate Buffered Saline with 0.1% Tween 20 (PBST). Tissue was then permeabilized with 10 µg/ml Proteinase K (Roche) in PBST for 10 min, washed twice in PBST for 2 min, post-fixed in fresh 4% PFA for 10 min, washed twice in PBST for 2 min, acetylated in 0.1 M triethanolamine (Sigma) containing 0.25% fresh acetic anhydride (Sigma) for 30 min, and washed three times in PBST for 10 min. For frozen section ISH, frozen tissue sections were submerged in room temperature 4% PFA PBS, pH 9.5 for 1 hr, washed twice in PBS pH 7.4 for 3 min then acetylated as above. Prior to hybridization, sections were pre-hybridized at 65°C for 1.5 hrs in 250 µl of hybridization buffer (50% distilled formamide, 5X SSC, 1% SDS, 50 µg/ml yeast tRNA, 50 µg/ml heparin sodium salt) then transferred into 250 µL of hybridization buffer containing 0.5 ng/µl of probe under Cover Well™ hybridization chambers (GraceBiolabs, Bend, OR) at 65°C for 18 hrs. Unbound probe was removed by washing like slides sorted by individual probe twice in 1X SSC for 15 min at room temperature, followed by 0.5X SSC for 60 min, at 65°C, and 0.5X SSC for 5 min at room temperature. Specific hybridization was then visualized via Digoxigenin specific IHC. For IHC, hybridized slides were equilibrated in maleic acid buffer, pH 7.5 (100 mM maleic acid, 150 mM NaCl, and 0.1% Tween 20) and non-specific antibody binding was blocked for 2 hrs in maleic acid buffer containing 1% non-fat dry milk. Equilibrated slides were then incubated in a 1
4,000 dilution of sheep anti-digoxigenin F(ab)2
alkaline phosphatase antibody (Roche) in blocking buffer overnight at 4°C. Unbound antibody was removed by extensive washing in maleic acid buffer for 15 min. Sections were next equilibrated in chromogenic buffer (100 mM Sodium Chloride, 100 mM Tris (Sigma, pH 9.5), 50 mM Magnesium Chloride Hexahydrate and 0.1% Tween 20) then exposed to 250 µl of chromogenic substrate (BM Purple AP Substrate, Roche) for times ranging from 1 day to one week with a daily change of substrate. Following sufficient deposition of blue/purple signal, slides were rinsed with water, counter-stained with filtered Nuclear Fast Red Stain (0.1% NFR in 5% aluminum sulfate, Kernechtrot, Germany), dehydrated through graded alcohols, cleared in xylene, and cover slipped in Permount (Fisher Scientific). A positive result is indicated by the presence of blue/purple precipitate on a pink/red background and amount of signal generated per unit of time can be used to estimate abundance of a targeted transcript. Using this approach, highly abundant transcripts (e.g. Gapdh
in tissue or cells or Il7
in pECFP-N1/IL-7 and pEYFP-N1/IL-7 transfected cells) are readily detected with 1 day (or less) of chromogenic exposure. Failure to detect signal after 7 days in combination with anticipated signals within technical controls is consistent with very low or absent mRNA expression and indicated by absence of blue/purple color on a pink/red background.
RNA extraction from whole thymus and real time RT-PCR
C57BL/6, Rag2−/− and K14 mouse thymi were harvested into RNA later (Ambion) and DNA-free total RNA was isolated using Triazol reagent (Invitrogen) via a modification to the provided protocol. Briefly, following addition of chloroform and phase separation, 50X DNase I buffer (1 M Tris, pH 7.0 and 100 mM MgCl2) was added to each aqueous phase to 1X final. One µl of Ambion DNase I (2 U/µl) was then added and incubated at RT for 15 min. Following completion of the standard protocol, concentration and purity of RNA yield was established by spectrophotometry (NanoDrop, NanoDrop Technologies, Wilmington, DE, USA) and quality confirmed by capillary electrophoresis (Agilent Bioanalyzer 2100 NanoChip).
Two microgram aliquots of each total RNA stock were converted into cDNA via hex primed reverse transcription (Thermoscript RT kit, Invitrogen). Each reaction was diluted with TE to produce a cDNA stock with a final volume of 100 µl and aliquots of each stock were then analyzed for relative amounts of Il7, Gapdh, and 18 s ribosomal RNA via Taqman Gene expression analyses (Applied Biosystems) using a Stratagene MX3000P thermocyler running MxPro software (ver 3.0). Ct values generated from each sample with the 18 s specific probe set were used to normalize expression of the two target genes (Il7 and Gapdh) using a ΔCt method with correction for variation in amplification efficiency. Normalized ratios were then used to determine the variance in target gene expression versus wild type, and the variance was then used to approximately calculate the corresponding ratio of each transcript to the other.
Cell preparation from thymus and cell sorting
Single cell suspensions were prepared from thymic tissue following published procedures 
. In brief, 10 thymi from C57BL/6 mice were harvested, punched with a scalpel, transferred to a beaker and stirred in 50 ml of RPMI 1640 (Mediatech Inc., Herndon, VA, USA) at 4°C for 30 min to remove the majority of the thymocytes. The resulting fragments were then digested in a series of three incubations in 5 ml of 0.125% (w/v) collagenase D/DNase I (Roche) with 0.1% (w/v) DNase I (Roche) in RPMI-1640 followed by a single digestion in 5 ml of 0.125% collagenase/dispase (Roche) with 0.1% (w/v) DNase I in RPMI-1640. Digestions were performed at 37°C for 15 min in an oven in tubes placed on the rotisserie for gentle agitation. Cells were collected after each digestion once thymic fragments had settled, pooled and kept on ice. After washing in PBS, cells were filtered through a 100 µm mesh and counted. Before staining, cells were depleted of hematopoietic cells using MACS CD45 MicroBeads (Miltenyi Biotec, Auburn, CA, USA) and autoMACS separation columns. After enrichment, cells were labeled using different combinations of the following antibodies: APC-CD45.2 (eBioscience), PE-CD11c (clone HL3, BD Pharmingen, San Jose, CA, USA), EpCAM (clone G8.8a) followed by secondary staining with PE-Cy5-anti-Rat IgG, FITC-Ly51(clone 6C3, BD Pharmingen), MTS-15 followed by secondary staining with FITC-ant-Rat IgG, and MTS-12 (anti CD31) followed by secondary staining with PE-anti-Rat IgG. Cell sorting was performed using a MoFlow Cytometer and cells were collected in RPMI-1640, washed and resuspended in lysing solution (RNAqueous-Micro Kit, Ambion, Austin, TX, USA).
RNA extraction from sorted stromal cells and absolute quantification of Il7 mRNA by real time RT-PCR
RNA was extracted using RNAqueous-Micro Kit (Ambion) following manufacturer's protocol. After extraction, 100 ng of RNA were retrotranscribed using Superscript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) and following manufacturer's instructions.
Specific primers for real time PCR quantification of Il7
mRNA were designed using Primer Express software (Applied Biosystems, Forster City CA, USA) and optimized for amplification and minimal primer dimer formations. Primers and probes were designed over a conserved region of the genome and synthesized by Applied Biosystems. Accurate quantification of Il7
mRNA was accomplished using a plasmid pcDNA3 vector containing the murine Il7
cDNA (a gift from J. Bream, Johns Hopkins University). DNA sequencing from both the 5′ and 3′ ends verified the identity. The real time PCR assays for Il7
plasmid DNA/cDNA were carried out in 10 µl reactions using Il7
specific primers with TaqMan Universal master mix and run on the ABI Prism 7900 (Applied Biosystems, Forster City CA, USA) (50°C for 2 min, 95°C for 10 min followed by 45 cycles at 95°C for 15 sec, 60°C for 30 sec). The murine glyceraldehyde-3-phoshate dehydrogenase (Gapdh
) plasmid DNA used as a standard was purchased from Serologicals (Gaithersburg, MD, USA). The real time PCR assay for Gapdh
plasmid DNA was carried out in 10 µl reactions using the murine Gapdh
control kit (Applied Biosystems) and run as described above. A standard curve was generated by plotting the log10
target dilution, of murine Gapdh
control template and murine Il7
on the X-axis against the cycle threshold (Ct
) value from serial dilutions (6 log dynamic range) of murine Gapdh
target DNA on the Y axis. Sensitivity and linear dynamic range were checked on the serial dilutions (10–106
copies/reaction) of Il7
plasmid DNA (a standard curve equation Y
−Mx+b is applied to identify the number of molecules of Gapdh
present in unknown samples). The Il7
PCR efficiency was 0.968 with a slope of −3.2, while the Gapdh
PCR efficiency was 0.975 and the slope was −3.3. The Il7
mRNA expression was then normalized to the Gapdh
expression values of the same unknown samples to quantify the absolute expression of all samples in the experiment.
Construction of the IL7promECFP mice by BAC recombineering
A bacterial artificial chromosome (BAC) containing the mouse Il7 gene (RP23-32I7, clone length 228391 bps) was obtained from Invitrogen. BAC DNA was purified using the Nucleobond BAC kit (Clontech) according to manufacturer's instructions and characterized by SpeI fingerprinting.
The construction of the IL7-ECFP-BAC was done using galK selection as described by Warming et al. 
. Briefly, a galK
cassette with homology to the first translated exon of Il7
(exon 1) was amplified using Expand High Fidelity (Roche), pgalK as template, and the following primers (ATG starting site of Il7
is in bold, sequences priming the pgalK plasmid are underlined):
IL7->galK F: 5′-CCT GCT GCA GTC CCA GTC ATC ATG ACT ACA CCC ACC TCC CGC AGA CCA TGC CTG TTG ACA ATT AAT CAT CGG CA-3′
and IL7->galK R: 5′-TCC CCG GCG CGC TAG GCG CAC CTA CTT GTG CGC ACC AGA GAG CAG CGC TTT CAG CAC TGT CCT GCT CCT T-3′
. PCR conditions were 94°C 15 sec, 60°C 30 sec and 72°C 1 min for 25 cycles. The PCR reaction was treated with DpnI and gel purified. Purified PCR product was transformed into heat-shocked and electrocompetent SW102 containing RP23-32I7 and Gal+
colonies were selected as described. Insertion of galK
resulted in a deletion of the remainder of exon 1 as well as deletion of the splice donor of intron 1. Next, the galK
cassette was replaced with a PCR product containing homology arms identical to the homology arms used in the first step, amplified from pECFP-1 (Clontech) using the following primers and PCR conditions as described above: galK->ECFP F: 5′-CCT GCT GCA GTC CCA GTC ATC ATG ACT ACA CCC ACC TCC CGC AGA CCA TGG TGA GCA AGG GCG AGG A-3′
and galK->ECFP R: 5′-TCC CCG GCG CGC TAG GCG CAC CTA CTT GTG CGC ACC AGA GAG CAG CGC TTG CCT TAA GAT ACA TTG ATG AGT TTG GA
. This PCR reaction product was DpnI-treated, gel-purified, and transformed into SW102 Gal+
cells and DOG resistant colonies were selected as described 
. One out of 10 analyzed BAC clones was correctly targeted after galK counter selection, and the modified area of the BAC was confirmed by direct BAC sequencing of large-prep BAC DNA using ABI PRISM BigDye Terminators (Applied Biosystems). The IL-7-ECFP BAC construct was linearized with PiSceI (New England Biolabs) and microinjected into fertilized ova of C57BL/6 females at the pronuclear stage. Mice were screened for acquisition of the transgene by Southern Blot Analysis of DNA obtained from tail biopsies using standard procedures. Genomic DNA was digested with BglII and analyzed with a 299 bp probe amplified from the RP23-32I7 BAC. The wild type band is 2.6 kb and the transgenic band is 3.6 kb.
Genotyping and selection of homozygous IL7promECFP transgenic mice
Genomic DNA from mouse tail biopsies was extracted by ethanol precipitation after digestion in digestion buffer (50 mM Tris-HCl, pH 8, 100 mM EDTA, 100 mM NaCl, 1% SDS) with 500 µg Proteinase K (Roche Applied Science, Indianapolis, IN, USA) overnight at 55°C and resuspended in 100 µl of 0.1X SSC buffer. Approximately 10 ng of this DNA was analyzed in a 20 µl SYBR green PCR reaction containing 1X Brilliant SYBR Green QPCR Master Mix (Stratagene), 30 nM ROX passive reference dye, and 300 nM each primer. The ECFP transgene was assayed using specific primers, and differences in input DNA were normalized using an endogenous reference gene, Folh1, in separate wells. Primer sequences used were CFP-F 5′-ATG CCA CCT ACG GCA AGC TG-3′, CFP-R 5′-TTC TGC TGG TAG TGG TCG GCG-3′, FolH1-F 5′-CCA AGC AGC CAC AAC AAG TA-3′ and FolH1-R 5′-TCC ATA GGG ATT TTG TGA TTC TG-3′. Real time PCR was performed in triplicate on a MX3000P Stratagene (Cedar Creek, TX, USA) instrument running an initial enzyme activating step of 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec. Normalized data was then analyzed using the −2ΔΔCt method to determine relative fold change compared against a known hemizygous animal (calibrator).
Immunohistochemistry and immunohistology
All tissue samples were fixed in 4% PFA for approximately 16 hrs at 4°C, then transferred to 20% sucrose and incubated for another 16 hrs at 4°C. Finally, tissues were blotted to remove excess sucrose, frozen into OCT compound (Sakura, 4583), and held at −80°C until sectioning. Seven µm sections were cut using a cryostat and stored at −80°C until the day of staining.
For IL-7 immunohistochemistry, sections were warmed to room temperature for approximately 60 min, then rinsed in 1X PBS for 3 changes, 3 min each. Antigen retrieval was performed using a citrate based buffer pH 6 (Biogenex, HK086-9K) in Milestone RHS-1 microwave processor for 10 min to reach 100°C, followed by 10 min at 100°C. Peroxide block was applied at 0.6% in 0.1% saponin (Sigma, S4521)/1X PBS (wash buffer) at room temperature for 15 min followed by rinsing in wash buffer 3 times, 3 min each. Anti-mouse IL-7 antibodies (rabbit polyclonal IgG from Santa Cruz, goat polyclonal IgG from Santa Cruz and R&D, monoclonal rat IgG2b from R&D, biotinylated rabbit polyclonal IgG from PeproTech, monoclonal mouse IgG from Amgen) were applied at different dilutions, in wash buffer, at room temperature for at least 60 min. Samples were then rinsed using wash buffer 3 times, 3 min each. Avidin-Biotin Complex reagent was applied (ABC, Vector) following manufacturer's instructions diluted in wash buffer, and incubated at room temperature for 30 min, followed by another rinse for 3 times, 3 min each with 1X PBS. Slides were then developed in DAB (Sigma, D5905-100TAB), counterstained, dehydrated, and cover slipped.
For ECFP-target cell identification and co-localization, tissue sections were warmed to room temperature for approximately 60 min, placed in acetone for 10 min at room temperature, and then dried at room temperature for approximately 5 min. Sections were re-hydrated in 1X PBS for 10 min followed by application of 2% normal goat serum/PBS (Vector, S-1000) for 20 min at room temperature. Serum in excess was removed (not rinsed) and primary antibodies were applied for 60 min at 37°C (unless differently specified) as a cocktail diluted in 0.1% BSA/1X PBS. For examining ECFP expression, anti-GFP (Abcam) was used at a 1
1000 dilution. Other antibodies were used at the following dilutions: FITC-anti-Ly51 (BD Pharmingen) at 1
1000; anti-G8.8a (gift from Gray D.) at 1
1000; anti-MTS-15 (gift from Gray D.) 1
20; biotinylated-CD45.2 (eBioscience) at 1
500; FITC-CD11c at 1
1000; CD31 (Santa Cruz) at 1
200 O/N at 4°C. The sections were then rinsed in 1X PBS for 10 min and corresponding secondary antibodies were applied as a cocktail, diluted 1
300 in 1% BSA/1X PBS, for 30 min at room temperature. The following secondary antibodies have been used: donkey anti-rabbit 488 (Molecular Probes), goat anti-rabbit 546, and biotinylated goat anti-rabbit (Vector) followed by streptavidin conjugated Alexa 546 (Molecular Probes) for anti-GFP; goat anti-rabbit 488 (Molecular Probes) for G8.8a; goat anti-rat 546 (Molecular Probes) for MTS-15; streptavidin conjugated Alexa 546 (Molecular Probes) for CD45.2; donkey anti-goat 546 (Molecular Probes) for CD31. Slides were rinsed in 1X PBS for 10 min, wiped dry, and cover slipped with Prolong Gold (Molecular Probes).
All samples were visualized with a Nikon Eclipse 80i microscope under consistent illumination and exposure conditions for each respective stain. Brightfield images were captured using Nikon DXM1200F digital camera and Nikon ACT-1 software. Fluorescent images were obtained using Exfo X-Cite 120 excitation, Nikon UV-2E/C, B-2E/C and G-2E/C filter cubes, Qimaging Retiga 2000R digital camera, and Media Cybernetics Image-Pro plus v5.1 software.
For anti-keratin staining, intact thymic lobes were fixed by immersion in 4% PFA, cryoprotected with phosphate buffered saline containing 30% (w/v) sucrose. Samples were then embedded in OCT (Sakura Finetek U.S.A., Inc., Torrance, CA, USA), frozen, and then sectioned at 5–7 µm with a cryostat. Sections were collected on SuperfrostPlus slides (Fisher Scientific, Pittsburg, PA, USA). After drying overnight, sections were hydrated in PBS and then incubated in a mixture of primary antibodies. To Troma1 hybridoma supernatant (Developmental Studies Hybridoma Bank, University of Iowa; dshb.biology.uiowa.edu/), was added goat anti-green fluorescent protein (Rockland, Gilbertsville, PA, USA), and different rabbit antibodies, either anti-keratin 5, anti-keratin-8 or anti-keratin 14 (all from Covance, Berkley, CA, USA). Controls consisted of mixtures of normal goat, rabbit, and rat IgGs diluted to equivalent concentration. Primary antibodies were applied for 1 hr at room temperature. After repeated washes in PBS, slides were incubated with a mixture of conjugated secondary antibodies diluted in PBS containing 10 mg/ml bovine serum albumin and 10% (v/v) normal donkey serum. Secondary antibodies were donkey anti-goat IgG Alexa 488, donkey anti-rabbit IgG Alexa 555, and chicken anti rat IgG Alexa 647 (all from Invitrogen-Molecular Probes, Eugene, OR, USA). Following incubation for 1 hr. protected from light, the slides were repeatedly washed with PBS, then incubated in 10 mM acetate buffer, pH 6, containing 1 mM CuSO4 for 10 min before a final wash in PBS. Coverslips were mounted with Fluoromount G (SouthernBiotech, Birmingham, AL, USA). Sections were viewed with a Leica microscope equipped with a digital camera (Orca-ER, Hamamatsu Photonic Systems, Bridgewater, NJ, USA) to collect images. Resulting monochrome images were converted to RGB images with Photoshop (Adobe, San Jose CA, USA).
Intravital multiphoton microscopy
Lymphocytes were isolated from OT-IxRAG−/−
mice and differentiated into central memory T-cells (TCMs) by stimulation with OVA peptide (SIINFEKL, a generous gift T.Mitchell, U.Louisville) followed by culture in the presence of IL-15 for 5 to 7 days 
. Differentiation of cells into TCMs was evaluated by FACS analysis for CD8, CD44, CD62L, CD122 and CCR7 expression. Intravital microscopy of mouse bone marrow was performed using a protocol modified from a previous report 
. Twenty-four hrs after i.v. injection of CFSE-labeled TCMs, mice were anesthetized with isoflurane (Baxter, 2.5% vaporized in an 80
20 mixture of O2
and air), and the hair in the neck and scalp was removed with hair removal lotion (Nair, Carter Products, NY). The frontoparietal skull was exposed and the mouse head was immobilized in a custom stereotactic holder. The imaging system was an LSM510 NLO Meta (Carl Zeiss, Jena, Germany) driven by a Chameleon femtosecond pulsed laser (Coherent Inc., Santa Clara, CA) tuned to 880 nm, and an inverted microscope (Axiovert 200; Carl Zeiss) equipped with a 40X water immersion objective (Achroplan IR, NA 0.8; Carl Zeiss). The microscope was enclosed in an environmental chamber in which anesthetized mice were warmed by heated air. Fluorescent cells were detected using a bandpass emission filter at 480/40 nm (for ECFP) or 525/50 nm (for CFSE). Vessels were visualized after i.v. injection of 70 kDa Texas Red conjugated-dextran (620/60 nm filter). Image stacks were collected with a 3 µm vertical step size at a depth of 100–150 µm below the skull bone surface. For 3D videos, 4 sequential image stacks were acquired at 3 mm z spacing to cover a volume of 154 µm×154 µm×9.0 µm with a 1 min interval between repetitive image stack collections. Imaging data were processed with Imaris (Bitplane, Zurich, Switzerland).