, 2002) Although the cell wall binding domain might be essential

, 2002). Although the cell wall binding domain might be essential for the enzyme’s lytic activity, some endolysins were reported to exhibit higher antibacterial activity after removal of their C-terminal domains (Borysowski selleck inhibitor et al., 2006). The mechanism of cell wall substrate recognition and the specificity and binding ability of the endolysins have been studied. Significant progress has been made using endolysins linked with green fluorescent protein (GFP). The specific

binding of endolysins to the cell wall substrate has been visualized by fluorescence microscopy (Loessner et al., 2002; Low et al., 2005; Korndoerfer et al., 2006; Briers et al., 2007). Corynephage BFK20, a lytic phage of the industrial producer

Brevibacterium flavum CCM 251, is the first corynephage whose genome was completely sequenced and analyzed (EMBL accession no. AJ278322) (Bukovska et al., 2006). Using a bioinformatics approach, three potential lytic genes in one cluster were identified on the BFK20 genome. In this study, we characterized BFK20 endolysin (gp24′) in detail. We have confirmed the two-domain structure 3-Methyladenine mouse of this endolysin. The catalytic and cell wall binding domains were separately cloned, isolated and characterized. The biological activities of BFK20 endolysin and its catalytic domain were demonstrated. The C-terminal cell wall binding domain appears to be unrelated to any of the previously known cell wall binding domains. Amino acid sequences of endolysins were searched using blastp (Altschul et al., 1997) on the nonredundant database using the sequence of gp24′ as the query. We selected those sequences with E-values over 9e−07 and one sequence from Corynebacterium diphtheriae NCTC 13129 with an E-value 3e−04. These sequences were aligned using clustalw2 (Thompson et al., 1994) and manually adjusted. A domain search was performed against the Pfam databases (Bateman et al., 2004). The bacterial strains used in this study were B. flavum CCM 251 (an l-lysine production strain), B. flavum strains ATCC

21127, 21128, 21129 and 21474, Brevibacterium Linifanib (ABT-869) lactofermentum BLOB (a mutant derived from B. lactofermentum ATCC 21798) (Santamaria et al., 1984), Corynebacterium glutamicum RM3 (Schäfer et al., 1990) and Bacillus subtilis wt PY79 (Youngman et al., 1984). Escherichia coli XL1 Blue (Stratagene) was used for cloning experiments and E. coli BL21(DE3) (Novagen) was used as a host for the expression of recombinant proteins. Escherichia coli strains were grown at 37 °C, and corynebacteria and bacilli were grown at 30 °C in Luria–Bertani medium (Sambrook & Russel, 2001). Corynephage BFK20 was propagated on B. flavum CCM 251 according to Bukovska et al. (2006). BFK20 phage particle isolation and phage DNA purification were performed according to Sambrook & Russell (2001).

Understanding the way in which hosts and pathogens interact began

Understanding the way in which hosts and pathogens interact began to unravel some of the mysteries of infection and disease. This led to the concept of

‘natural immunity’ to infection, which was indispensable for vaccine design. In 1908, Metchnikoff was awarded the Nobel Prize in Medicine jointly with Paul Ehrlich for their work on the theory of immunity. At the end of the 19th century, many of the fundamental aspects of vaccinology were in place because INCB024360 nmr of the pioneering work of scientists like Pasteur, Koch, Metchnikoff and Ehrlich. The most important advance was the demonstration that the administration of pathogens, either attenuated or inactivated, resulted in protection against the disease caused selleck kinase inhibitor by the respective native pathogen. Developments

in pathogen attenuation processes led to consistent production of attenuated microbes, and many of the vaccines employed today are still based on these developments. Figure 1.8 shows the various vaccine technologies developed over time. At the end of the 19th century, Émile Roux and Alexandre Yersin discovered that diphtheria and tetanus bacilli produce soluble molecules called exotoxins, which caused the symptoms of these infections. Soon after this discovery, Emil von Behring and Shibasaburo Kitasato postulated the serum antitoxin concept. The use of the term ‘immunisation’ dates from this work, referring to the rabbit serum that contained the antitoxin as immune serum. First Nobel Prize in Medicine The discovery of antibodies in 1890 and passive immunotherapy of diphtheria was honoured in 1901 when the first Nobel Prize in Medicine was awarded to Emil von Behring. In 1924, Gaston Ramon, a veterinarian at the Pasteur Institute, applied chemical inactivation to bacterial toxins to produce toxoids of diphtheria and tetanus. By this method, he transformed the

tetanus toxin with formaldehyde and heat into a safer, non-toxic product, without changing its immunogenic potential. He called this chemically treated product ‘anatoxin’ (ie toxoid). This discovery was also applicable to the toxin produced by the diphtheria bacillus. The diphtheria toxoid produced by Oxymatrine this method was used in a vaccination programme to greatly minimise fatal cases of diphtheria in infants. The tetanus toxoid vaccine was widely used to prevent tetanus from battle wounds sustained during World War II. The introduction of tetanus vaccination has almost eliminated the number of cases in developed countries; however, tetanus remains a problem, largely in the developing world ( Figure 1.9). Worldwide annual deaths in 2004 from tetanus were estimated to be 163,000, 144,000 of which occured in children less than 5 years of age ( WHO, 2009).

The association of exenatide and sitagliptin with pancreatitis wa

The association of exenatide and sitagliptin with pancreatitis was documented since 2006 and prompted close monitoring [14] and [15]. Later, the potential risk appeared to be increased by diabetes per se; post-approval studies have documented cases associated with incretin use, but a causal relationship between treatment and pancreatitis was neither proved nor excluded [16], [17], [18], [19] and [20]. In the registry, a few additional reports of non-severe pancreatitis or simply raised levels of pancreatic enzymes were also recorded, without differences

between drugs. When these non-adjudicated ADRs were summed up to severe pancreatitis, the total incidence Venetoclax cost of pancreatic events was in the range reported in the general population with diabetes and should be considered in the context of the notoriety bias generated by alerts. A 2013 comprehensive review of preclinical and clinical data on pancreatic safety by the European Medicines Agency concluded that the concerns on the risk of pancreatitis

should not be minimized [21]. Later, the publication of two large cardiovascular outcome DPP-Is trials [13] and [22] and epidemiological data [23] stifled the debate; a 2014 joint Food and Drug Administration (FDA)–European Medicines Agency (EMA) assessment concluded with a low-risk [24] but suggested continuous Dabrafenib capture of data. As expected, exenatide and DPP4-I add-ons to metformin were accompanied by low rates of hypoglycemia [25]. On the contrary, a two-to threefold increase in hypoglycemia was observed in combination with sulfonylureas, both with and without metformin, but very few cases were recorded as severe ADRs, requiring PJ34 HCl hospital admission. These data are in keeping with registration studies and with recent clinical trials showing that DPP4-Is are associated with very low rates of hypoglycemia when combined with metformin

[26], despite similar or only moderately inferior glucose-lowering efficacy compared to sulfonylureas. The analysis of discontinuation rates and metabolic effects may give hints for an appropriate use of these drugs in the community. This approach seems sound, as confirmed by a sensitivity analysis in a subset of selected centers with adherence to follow-up ≥80% (Supplementary Tables 1 and 2). As expected, the discontinuation rates of all drugs increased systematically with higher baseline HbA1c. They also increased with age for exenatide, not for gliptins, indicating a preferential use of oral agents in elderly subjects for whom a less strict metabolic target may be preferred [3], [4] and [27]. On the contrary, weight loss might be the reason for the lower discontinuation rates of exenatide with increasing BMI, despite injections and higher baseline HbA1c. Two subpopulations, with limited safety data in registration studies, deserve particular attention.

Eighteen-week-old male Swiss mice were supplied by the Animal Hou

Eighteen-week-old male Swiss mice were supplied by the Animal House of the School of Pharmaceutical Sciences and Chemistry Institute

from University of Sao Paulo. The animals were fed a standard pellet diet and water ad libitum, and before each experimental procedure, the animals were anesthetized with ketamine/xylazine solution (80 mg/kg; 8 mg/kg; i.p.). All procedures were performed according to the Brazilian Society of Science of Laboratory Animals (SBCAL), for proper care and use of experimental animals and approved by the local ethics committee (process GDC-0199 clinical trial number 196). Five mice were randomly placed in an exposure box and exposed to aerosolized HQ at concentrations of 12.5, 25 or 50 ppm or vehicle (saline solution with 5% ethanol) for 1 h, once a day, for 5 days. An ultrasonic nebulizer (NS®, Sao Paulo, Brazil) was used to nebulize the solutions in the box. According to the manufacture’s information the particle size generated Selleck Inhibitor Library by the nebulizer is within the range 0.5–10 μm. Two openings at the opposite side of the chamber, relative to the introduction of solutions, allowed the air to seep out. This process was performed in an exhaust hood. It is important to emphasize that concentrations of HQ employed in the current study were lower than those

established for in vivo exposure in the literature ( NIOSH Guideline, 1988 and IPCS-INCHEM, 1994). A dose–response

effect had been previously performed and 5-days exposure was the shortest period to evoke the toxic effect (data not shown). HQ concentrations in the exposure box were determined according to NIOSH, protocol No. 5004. The induction of pulmonary inflammation was performed 1 h after the last vehicle or HQ exposure using a similar exposure box approach. LPS (0.1 mg/ml) was aerosolized for 10 min at a Non-specific serine/threonine protein kinase rate of 1 ml/min. Three hours after LPS inhalation, the animals were anesthetized and arterial blood was collected from the abdominal aorta. The total and differential counts were performed as previously described (Macedo et al., 2006). BALF was collected from vehicle- or HQ-exposed animals to determine the number of migrated leukocytes and concentrations of cytokines as previous described by De Lima et al. (1992). MPO activity was determined in the lung tissue obtained from vehicle- or HQ-exposed animals accordingly to Bradley et al. (1982). Lung of vehicle or HQ exposed mice were surgically removed, frozen in nitrogen–hexane solution, cryosectioned (8 μm thickness) and fixed in cold acetone (10 min). Briefly, sections were incubated overnight with Superblock solution to avoid nonspecific binding.

We thank the patients and their families,

We thank the patients and their families, PARP inhibitor the study site coordinators and nurses, all of whom made this study possible. Raymond Mankoski, M.D., Ph.D., Gerald Cox, M.D., Ph.D., and Lisa Underhill, M.S. of Genzyme, a Sanofi company

reviewed and contributed to this manuscript. Laurie LaRusso, Chestnut Medical Communications, provided medical writing support, which was funded by Genzyme. The study was supported by research funding from Genzyme to E.L., N.W., M.D., G.M.P., E.A.A., H.R., and A.Z. Authorship contributions M.J.P. designed the study; E.L., N.W., M.D., G.M.P., E.A.A., H.R. and A.Z. recruited patients and conducted the study research; J.A. performed the statistical analyses; M.J.P., A.C.P., and BAY 80-6946 L.R. analyzed and interpreted the results and wrote the manuscript. All authors reviewed early and final drafts of the manuscript and were fully responsible for the content and

editorial decisions related to this manuscript. Role of the funding source This trial was funded by Genzyme, a Sanofi company. The Genzyme project team developed the design and set-up of the trial in collaboration with study investigators and regulatory authorities. Study data were monitored by clinical research associates contracted to Genzyme in each study region. Analyses were performed by the Genzyme Biomedical Data Science and Informatics division. All authors had access to the study data. An independent Data Monitoring Committee (DMC) provided additional oversight Glycogen branching enzyme of patient safety through periodic and ad-hoc reviews of study data, and review of information on patient discontinuations/withdrawals. Genzyme provided funding for medical writing services. The decision to submit the manuscript for publication was made jointly

by all authors. “
“Breast cancer is the most common cancer in women and the second most common cancer worldwide [1]. In the last decade, targeted therapy in breast cancer has become part of routine clinical protocols all over the world. Trastuzumab, a humanized monoclonal antibody that targets human epidermal growth factor receptor 2 (HER2), is routinely used to treat patients with breast carcinoma who overexpress HER2 [2] and [3]; when combined with chemotherapy in the metastatic setting, trastuzumab improves progression-free survival and overall survival by years [4]. Other HER2-targeting drugs (e.g., the kinase inhibitor lapatinib [5], the antibody pertuzumab [6], the antibody–drug conjugate ado-trastuzumab emtansine [T-DM1] [7]) have been approved for use in the treatment of HER2-positive metastatic breast cancer. At the same time, it has been shown that lapatinib (when added to paclitaxel) [8] and pertuzumab (as a single agent) [9] offer no clinical benefit to patients with HER2-negative metastatic disease.

05 < χ20 05,1 = 3 84) Large populations were investigated in F7,

05 < χ20.05,1 = 3.84). Large populations were investigated in F7, RHL-F2 and RHL-F3 with 179, 720 and 7400 medium grain individuals found in the total populations of 800, 3000, 30,000 individuals, respectively. Likewise, the segregation ratios

of big versus medium grain fit to a ratio of selleck chemicals llc 3:1 (χ2 = 2.80, 1.55, 1.76 < χ20.05,1 = 3.84). A total of 129 polymorphic markers were detected between R1126 and CDL from 400 SSR, SFP and ILP markers, and 113 well-distributed polymorphic markers were used to survey the ten medium-grain plants, ten big-grain plants of F7 population and parents. The GS2 gene was roughly mapped to the interval between RM13819 and RM13863 on the long arm of chromosome 2. We found that six SSR markers, namely RM3289, RM1342, RM5305, RM13819, RM3212 and RM13863, located on chromosome 2 were clearly associated with the medium-grain phenotype. After further studying 179 F7 medium-grain plants using these six markers, the GS2 gene was located between RM13819 and RM13863 with genetic distances of 0.84 cM and 0.28 cM, respectively. Furthermore, 0 recombinant was detected by marker RM3212. These data were derived according to the recombinants revealed by each marker, covering a ~ 553-kb physical segment on the region

of rice chromosome 2 ( Fig. 2-A). Z-VAD-FMK mouse To fine-map the GS2 locus, 29 polymorphic InDels were selected from 142 InDels developed according to the information

on the sequence (R1126 and Nipponbare) between RM3212 and RM13863. Further genotyping 2576 medium grain plants of the RHL-F3 revealed one recombinant in the proximity of GL2-35-1 and GL2-12. In addition, RM3212 and GL2-11 were verified to be linked to the GS2 gene. The GS2 locus was therefore Rucaparib finally narrowed down to the genomic region flanked by GL2-35-1 and GL2-12, a fragment of approximately 33.2 kb in length ( Fig. 2-B). In the 33.2-kb genomic interval of the Nipponbare genome, a total of three putative genes including LOC_Os02g47280, LOC_Os02g47290 and LOC_Os02g47300 were predicted by TIGR rice annotation (http://rice.plantbiology.msu.edu/cgi-bin/gbrowse/rice/) (Fig. 2-B). LOC_Os02g 47280 encoded a putative growth-regulating factor; LOC_Os02g47290 and LOC_Os02g47300 encoded hypothetical proteins with no further evidence such as expressed sequence tag (EST) or RNA. Because of the recent developments in bioinformatics and genome sequencing to yield an impressive number of molecular markers, many major QTLs responsible for grain shape and yield have been fine mapped and cloned in the past 20 years. In this paper, we fine mapped GS2 using RHL population developed from a big-grain rice line CDL and a medium-grain line R1126. GS2, which controls grain length and width, was narrowed down to a candidate genomic region of 33.

The few studies that examined actual evapotranspiration reported

The few studies that examined actual evapotranspiration reported that actual

evapotranspiration would increase over the TP generally but with spatial variations (Yang et al., 2011, Zhang et al., 2007a and Zhang et al., 2007b), and the result would be less available water for streamflow. Cuo et Dabrafenib al. (2013a) looked at the impacts of actual evapotranspiration change on streamflow and found that increases in actual evapotranspiration were larger during May–October when compared to the other months. The same authors noted that actual evapotranspiration change was the second most important factor besides precipitation change in causing the annual and seasonal streamflow decreases in YLR. The difficulty in obtaining existing MS-275 chemical structure hydrological observations collected and maintained by the Chinese Ministry of Water Resources and the local bureaus of water resources due to their data policies and the harsh environment unfavorable for setting up and maintaining hydrological observational sites on the

TP pose great challenges for hydrological research in the region. Overcoming these challenges requires sustained and coordinated efforts from all levels of agencies and researchers alike. In addition, there are other hydrological research topics on the TP that need to be addressed. Among them, three most important scientific issues are discussed below. Climate systems dictate precipitation and temperature on the TP, which in turn regulate streamflow. Large-scale atmospheric systems such as the mid-latitude westerlies, East Asia and Indian monsoons, North Atlantic Oscillation, mafosfamide Arctic Oscillation, ENSO and local circulations all play roles in affecting the weather and climate of the TP (Tian et al., 2007, Cuo et al., 2013b, Yao et al., 2013 and Gao

et al., 2014). As an example, Wang et al. (2006) showed that above-average annual precipitation in YLR and YTR is caused by enhanced moisture transport by the Indian monsoon when Mongolian low pressure and the westerlies are weak. Li et al. (2007) reported that above normal precipitable water vapor is transported to TRB by the intensifying westerlies as the northerlies become weakened. Any changes in precipitation would have strong implications for streamflow in the basins. Relating streamflow to climate system indices could potentially reveal the impacts of the climate systems on streamflow and help understand the spatial and temporal changes of streamflow over the TP. Ding et al. (2007) compared the annual streamflow changes among YLR, YTR and BPR and found that the changes were out of phase between YLR and BPR, and they attributed that to the differences in the prevailing systems.

24 The current study uses a prospective cohort of initially uninf

24 The current study uses a prospective cohort of initially uninfected households with active case finding. This is considered to be the gold standard design for influenza household studies and should provide a relatively representative and unbiased description of transmission and shedding dynamics.25 The participants in this

study had been enrolled in the cohort since December 2007 and most had blood samples collected and tested by serology just prior to the pandemic such that prior immune status and susceptibility could be confirmed. The research was approved by the institutional review board of the National Institute of Hygiene and Epidemiology, Viet Nam, the Oxford Tropical Research Ethics Committee, University of Oxford, UK. All participants provided written informed consent. The investigations described here were conducted as part of an ongoing household-based influenza cohort study that has been Neratinib ic50 described in detail elsewhere.26 In brief, households from a commune in Ha Nam Province, in northern Viet Nam were selected at random. 940 members click here of 270 randomly selected households were enrolled. Index cases were detected via active surveillance for influenza-like illness (ILI), defined as a fever >38 °C

and cough, or sore throat. Health workers examined all persons in confirmed A(H1N1)pdm09 case households, including those without symptoms, each day for up to 15 days during the first pandemic wave (September–December 2009). Examinations included collection of nose- and throat-swabs for quantitative RT-PCR and full-genome sequencing; mouth temperature measurement, scored on a 5-tier scale (36–36.9 = 1, 37–37.9 = 2, 38–38.9 = 3, 39–39.9 = 4, ≥40 = 5); and evaluation of symptoms (sore

throat, nasal congestion, Exoribonuclease runny nose, sneezing, dry cough, wet cough, headache, diarrhoea, myalgia, fever, and wheeze), which were scored on a 3-tier scale (none = 0, mild = 1, or moderate/severe = 2). A cough was defined as wet or productive if sputum or material from the bronchi was expectorated. Participants were also asked if they took the day off work because of illness or to care for another household member that was ill, and if they took oseltamivir. Blood samples were collected for serology in June 2009 and April 2010. Separate flocked swabs (Copan, Brescia, Italy) were used to firmly swab the entire posterior pharynx and tonsillar area and the nasal cavity at the level of the turbinates. Nasal and throat swabs were combined in 1 tube containing 3 ml of viral transport medium, and transferred to the laboratory within 24 h where they were vortexed before aliquoting and storing the media at −80 °C. RNA was extracted from swab media and assessed by real-time reverse-transcriptase polymerase chain reaction (RT-PCR), according to WHO/USCDC protocols (CDC reference no. I-007-05, http://www.who.int/csr/resources/publications/swineflu/CDCRealtimeRTPCR_SwineH1Assay-2009_20090430.pdf).

Brains were washed in phosphate-buffered saline (PBS) (with Ca++/

Brains were washed in phosphate-buffered saline (PBS) (with Ca++/Mg++) and meninges were thoroughly peeled off and discarded. White matter was carefully removed. The grey matter was collected in HEPES-buffered MEM containing 10% foetal calf serum (MEM-H 10% FCS), Venetoclax price forced through a 50 ml syringe to produce a slurry, and mixed with an equal volume of MEM-H 10%. Tissue was gently homogenised in a glass Wheaton Dounce tissue grinder (Jencons Scientific Ltd., Leighton Buzzard, UK) (89–127 μm clearance, 15 strokes;

25–76 μm clearance 15 strokes) and sequentially filtered, first through 150 μm nylon mesh, then through 60 μm nylon mesh. Microvessel fragments trapped on the 150 and 60 μm meshes were kept separate and digested at 37 °C for 1 h in medium M199 containing 10% FCS, 223 U/mg collagenase, 211 U/mg trypsin and 2108 U/mg DNase with continuous agitation. Microvessels were washed off the meshes with the enzyme mixture, centrifuged for 5 min at 240g at 4 °C to remove enzyme, then resuspended in MEM-H 10% FCS and centrifuged again; the resulting vessel fractions were kept separate as ‘150s’ and ‘60s’, the latter giving higher TEER. The ‘60s’ were used for all experiments described here. Digested fragments were resuspended in 10% DMSO in foetal calf serum, brought slowly to −80 °C and stored in liquid nitrogen. Six pig brains gave 12 1 ml aliquots of ‘60s’. Capillary fragments

were thawed and resuspended in plating medium consisting of DMEM with 10% BPDS with 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, 125 μg/ml heparin, with 4 μg/ml puromycin to kill contaminating

cells, especially pericytes (Perrière et selleck compound al., 2005). One aliquot was plated into two T75 flasks coated with lab-prepared rat tail collagen (Strom and Michalopoulos, 1982) and 7.5 μg/ml fibronectin, and grown to 70–80% confluence. Cells were detached by brief trypsinisation (500 BAEE units trypsin and 0.47 mM EDTA.4Na in HBSS without Ca2+ or Mg2+), then centrifuged at 360g for 5 min. The pellet of these first passage (P1) cells was resuspended selleck kinase inhibitor in plating medium containing DMEM, 10% BPDS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine and 125 μg/ml heparin. Cells were seeded onto collagen/fibronectin coated Transwell-Clear inserts at a density of 1×105 cells/cm2 or at 1×104 cells/well in 96-well plates for functional studies and grown for 2–3 day until confluent. The medium was changed to serum-free medium supplemented with 550 nM hydrocortisone ( Hoheisel et al., 1998) and the cells were treated with 250 μM pCPT-cAMP and 17.5 μM RO-20-1724 ( Rubin et al., 1991); these supplements helped to improve differentiation of BBB properties, especially tight junction maturation ( Förster et al., 2005). PBECs were used in experiments 24 h after this medium change. The quality of the model in terms of cell growth was assessed according to the time the cultures took to become confluent.

Nucleotide sequences were determined

Nucleotide sequences were determined 3-MA price using a Dye Terminator Cycle Sequencing kit (Applied Biosystems, Tokyo, Japan) and the ABI 3730xl DNA sequencer (Applied Biosystems). Sequences were compared with the National Center for Biotechnology Information (NCBI) nucleotide database using the Basic

Local Alignment Search Tool (BLAST) program. In situ hybridization was performed as described by Matsunaga and Okanoya (2008) and Kato and Okanoya (2010). Common marmosets were removed from the family cage, anesthetized with a mixture of ketamine and xylazine, and then killed by exsanguination. The marmoset could freely express calls before anesthesia. Brains were quickly dissected, frozen on dry ice in an embedding medium (Tissue-Tek; Sakura Finetek, Torrance, CA, USA), and then cut into 20-μm-thick coronal sections on a cryostat. Digoxigenin (DIG)-labeled probes were generated using the original cDNA PCR products as templates. PCR products were generated using universal primers (SP6 primer, 5′-TAATACGACTCACTATAGGG-3′; and T7 primer, 5′-TAATACGACTCACTATAGGG-3′) and purified using the Wizard® SV Gel and PCR Clean-Up System (Promega). Probes were synthesized using T7 or SP6 RNA polymerase (Roche Diagnostics, Switzerland) and a DIG-labeling mix (Roche Diagnostics). Selleckchem AC220 Glass-mounted brain tissue sections were fixed in 4% paraformaldehyde for 10 min

and then rinsed three times with phosphate-buffered saline (PBS) for 5 min each. Sections were acetylated for 10 min in distilled water containing 1.35% triethanolamine, 0.25% acetic anhydride, and 0.065% HCl, and then incubated in PBS containing 1% Triton X-100 for 30 min. Next, sections were rinsed three times with PBS for 5 min each time, and then incubated

in a hybridization solution of RNase-free water containing 50% formamide, 5 × saline sodium citrate (SSC), 5 × Denhardt’s solution, 0.24 mg/mL yeast tRNA, 0.5 mg/mL salmon sperm DNA, and labeled probes. The sections were coverslipped in a humid box and incubated overnight at 72 °C. The next day, coverslips were removed by placing sections in pre-warmed 5 × SSC at 72 °C, and then the sections washed by sequential incubations for 2 h at 72 °C in 0.2 × SSC, for 5 min at room temperature (RT) in Liothyronine Sodium 0.2 × SSC, and for 5 min at RT in buffer 1 (RNase-free water containing 0.1 M Tris–HCl (pH 7.5), 0.15 M NaCl, and 0.001% Tween 20). Sections were then incubated for 1 h in a blocking solution consisting of buffer 1 supplemented with 10% sheep serum. Next, sections were incubated overnight at RT with an alkaline phosphatase-conjugated anti-DIG antibody (1:5000 dilution in blocking buffer; Roche Diagnostics). The following day, sections were washed three times in buffer 1 for 5 min each at RT, and then incubated for 15 min in buffer 2 (0.1 M Tris–HCl (pH 9.5), 0.1 M NaCl, 50 mM MgCl2, 0.001% Tween 20, and 0.