IB10) mouse ES cells. After G418 (GIBCO) selection, clones were analyzed for homologous recombination by Southern blotting, hybridizing with external probes both at the 39 and 59 end as well as an internal probe. ES cells carrying the disrupted allele were microinjected into C57/BL6 blastocysts. Resulting chimeric mice were bred to derive animals heterozygote for the TGM3 mutation. These were subsequently either interbred to produce homozygous mice or repeatedly crossed onto a C57/BL6 background. Histology and immunofluorescence staining of tissues For histology, tissues were fixed in 4% paraformaldehyde, embedded in paraffin and 6 mm sections cut. Sections were stained with haematoxylin and eosin. For immunofluorescence microsco- py, either unfixed tissue, embedded in Tissue Tec (Sakura, Japan) and snap frozen in liquid nitrogen or paraffin embedded tissue was sectioned. After blocking, sections were incubated for 1 h at RT with rabbit polyclonal antibodies against loricrin, involucrin, filaggrin, keratin1, keratin10, keratin14 (Covance Research Products), and trichohyalin (a kind gift from Dr. Edward O’Keefe, University of North Carolina, USA), mouse monoclonal antibod- ies against TGase 1 (Paesel+Lorei) or rat monoclonal antibodies against TGase 2 (Neomarkers) and TGase 3 (a kind gift from Dr. Kiyotaka Hitomi, Nagoya University, Japan). Labeling was detected with goat antibodies against rabbit, mouse or rat immunoglubulins conjugated to Cy3 or Alexa 488. Sections were examined with an Axiophot microscope (Zeiss). Immunoblotting and protein detection Tissues of TGM3 mutant and control mice were homogenized in cold lysis buffer (20 mM Hepes, 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100 and 0.1% SDS), centrifuged at 16.0006 g for 30 min at 4uC and the supernatant was removed. For the extraction of hair protein, hairs were plucked from 2 week and 4 month old mice, immersed in 2% SDS and 5% b-mercaptoethanol overnight at 4uC and after boiling for 5 min centrifuged at 16.0006 g for 30 min at 4uC. Proteins in the supernatants were separated on an 8% SDS polyacrylamide gel and either stained with Coomassie brilliant blue or transferred to nitrocellulose. After blocking either with 5% nonfat milk or with 1% casein dissolved in TBS, the nitrocellulose membranes were incubated with primary antibodies. These were rabbit polyclonal antibodies against TGase 3 [16], loricrin, involucrin, filaggrin, keratin1, keratin10, keratin14, or trichohya- lin, and mouse monoclonal antibodies against TGase 3 (for sources see the section above). Mouse monoclonal antibodies were diluted in 1% casein/TBS while rabbit polyclonal antibodies were diluted in 5% nonfat milk/TBS. After probing with the primary antibodies for 1 h at RT and washing, membranes were incubated with horseradish peroxidase-conjugated swine antibodies against rabbit or mouse IgG (Dako) followed by chemiluminescence detection (ECL, Amersham). For identification of proteins, bands from Coomassie brilliant blue stained gels were excised, in-gel digested with trypsin and analyzed in a Bruker Reflex IV MALDI- TOF mass spectrometer. The identity was determined from the peptide masses using the Mascot search engine. Transglutaminase activity For transglutaminase activity assays, NN’dimethylcasein was coated onto microtitre plates, which were then blocked with BSA. Skin proteins were extracted by homogenization in 0.1 M Tris- HCl, pH 8.0 followed by clarification via centrifugation. In some cases dispase was added to activate zymogen forms of the enzyme. The supernatant was placed in the wells in the presence of 25 mM CaCl2 and 0.5 mM biotin-cadaverin (Molecular Probes) as co- In vivo Role of TGase 3 substrate. After incubation at 37uC, the reaction was stopped with EDTA. The wells were washed with 0.1 M Tris-HCl pH 8.0/ 0.01% Triton X-100 and incubated with Avidin-HRP for 1 h. After washing peroxidase activity was detected with ABTS (2,29- azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) in 0.03% H2O2 with the product measured OD at 405 nm. Hair Analysis Hair integrity was analyzed by microscopy after incubating hair overnight at 65uC with agitation in 50 mM sodium phosphate buffer, pH 7.9, containing 2% SDS and 20 mM dithiothreitol to destroy disulfide bonds. Preparation of corneocytes Corneocytes were isolated from 2 mm biopsy plugs from the ears of 4 week old female TGM3 mutant and control mice. The tissue was heated at 95uC for 30 min in 25 mM DTT and 2% SDS in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, and subse- quently centrifuged at 50006 g for 10 min. After discarding the supernatant and washing the pellets twice, corneocytes were suspended in 10 mM Tris-HCl, pH 8.0, containing 1 mM EDTA. The corneocytes where subjected to ultrasound for varying lengths of time using a cup horn sonicator (Misonix, Farmingdale). The corneocyte suspension was then examined for the presence of intact cells. Assays for barrier function To analyze potential barrier defects, Lucifer yellow penetration assays were performed. 1 mM Lucifer yellow solution was placed upon the backs of new born mice. After 1 h the mice were killed and frozen sections were prepared. Penetration of the dye was analyzed using a fluorescence microscope. For toluidine blue staining, newborn mice were killed and then dehydrated by incubations (1 min each) in 25%, 50%, and 75% methanol/PBS followed by 1 min in 100% methanol. The cadavers were then rehydrated with the same series of methanol solutions (1 min incubations), washed in PBS, and stained for 1 min in 0.0125% toluidine blue (Fluka) dissolved in PBS. After destaining in PBS, the mice were photographed. Transepidermal water loss from the ventral and dorsal skin of adult mice was determined using a Tewameter as described previously [14]. Wound Healing For wound healing experiments TGM3 deficient mice were back-crossed to C57/Bl6 mice for seven generations and then intercrossed to produce wild type and homozygous mutant litter mates. Wounds of 4 mm diameter were produced in anesthetized females by excising skin, the sub-cutaneous fat and the panniculus muscle with a biopsy punch (STIEFEL, Germany). Wounds were left uncovered and digitally photographed at the indicated time points. Macroscopic wound closure was determined by measuring the wound area and wound size was expressed as percentage of initial wound area. For histology, the complete wounds were excised with a margin of surrounding skin. Tissues were either fixed for 2 hrs in 4% paraformaldehyde before paraffin embed- ding or frozen unfixed in Tissue Tec. Serial sections were cut from the surrounding wound margin across the center of the wound towards the opposite wound edge in the caudocranial direction. The section with the largest wound diameter was defined as the wound center. Only sections from the wound middle were used for immunostaining. Antigen retrieval was performed by incubation with 0.1% trypsin for 10 min. at 37uC. PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e34252