When both are in agreement within the variation of the measured frequency shift, the assumed local current density, i(y), becomes the local current

density obtained from the measurement. This method is called inversion analysis. When measuring a NMR signal under conditions of a long TR   (>T  1 of 1H in a PEM, which is about 870 ms) and a short TE   (Bleomycin of the NMR signal, (SI2+SQ2)1/2, averaged over the time period “B” in Fig. 6. The relationship between the water content in the PEM and the intensity of the NMR signal were obtained from measurements at relative humidity levels from 30% to 85%. This relationship was used to make a calibration curve for converting the intensity of a NMR signal to the water content in the PEM. The time dependent change of the water content in the PEM measured by the method described above is shown in Fig. 9. As a typical result, the plots AZD6244 research buy measured in three positions in

the gas flow channel within the PEFC are shown in this figure; two of them were at the gas inlet and outlet, and the other was at the center position of the PEFC. In addition, in order to reduce the variation in the measurements, six signal intensities were averaged, and then shown in the figure as one plot. It is found from Fig. 9 that the water content at the up-stream side maintained a small value, and that the water content at the center varied greatly. The water drops which came out from catalyst layer of the MEA and contacted the planar

surface coil are observed as a very large NMR Ribose-5-phosphate isomerase signal. In this research, when a large NMR signal was measured exceeding the maximum water content of the MEA, it was considered that flooding arose. The spatial distribution of local electric current density calculated by the method described above is shown in Fig. 10 and shows the following phenomena. Since the electronic load equipment used was operated in constant current mode, the time dependent change of the local current generated in the PEFC was smaller than that of the water content. On the other hand, a difference in the spatial distribution of local current density arises. Because the concentrations of hydrogen and oxygen in the channels fall at the down-stream side (−y part) due to gas consumption, the local current density at the down-stream side (+y part) was relatively small compared with that at other locations. On the down-stream side, since the rate of generation of water became low due to the lower local current density, the change of water content in the PEM was small.

PAHs were also reported to be AR antagonists. The study indicated that these petrogenic

compounds are responsible for most of the ER and AR mediated activity in PWs. In summary, these studies document that compounds present in PW have a potential to exert endocrine effects in fish. The experimental exposure levels studied cover a range of PW concentrations that are typically found in close proximity to PW discharge points. They might therefore elicit Angiogenesis inhibitor effects on fish standing close to platforms. Meier et al. (2010) still concluded that widespread and long lasting xenoestrogenicity and reproduction effects of PW on the population level in fish are unlikely. This was also supported by Sundt et al. (2011) who compared data from PW-exposed fish in the laboratory to similar data from Atlantic cod caged at the Ekofisk oil field in the NS. No Vtg induction was observed in fish exposed experimentally to PW in the dilution range 0.125%–0.5% PW giving 2.6–11 mg L−1 AP metabolites in the fish bile. Levels of the corresponding APs in the water ranged from 3.0 to 9.7 μg L−1. In fish caged about 200 m from the large Ekofisk PW outfall (average rate 37 000 m3 day−1)

the AP metabolite levels were significantly elevated compared to control Selleck BGB324 cages, but still one order of magnitude lower than in bile from the lowest exposure concentration in the laboratory experiment. It was therefore not possible to determine a LOEC (Lowest Observable Effects Concentration) for AP metabolites from these studies. Since LOEC must be higher than the highest observed NOEC of 11 mg L−1 AP metabolites, and the AP metabolite levels

in the caged cod were only a fraction of this, the AP content in the Ekofisk PW discharge was well below Sinomenine a critical level for induction of Vtg. Still, the critical level for induction of Vtg is probably not far above these cited values, which is supported by Tollefsen et al. (2011) who found elevated Vtg levels in 72% of individual male Atlantic cod exposed to 21 μg L−1 of sum C1–C5 APs. Meier et al. (2011) showed that oral exposure to a mixture of 4 APs affected the endocrine system and gonad development in cod through changes in the hypothalamic-pituitary-gonadal (HPG) axis at doses that were much lower than those that resulted in Vtg induction. So, although Vtg is a sensitive parameter for detection of endocrine disruption, lack of response in Vtg alone does not exclude that the endocrine system in fish may be disturbed by PW components. Compelling evidence thus exists from in vitro bioassays that PW contains estrogenic compounds ( Thomas et al., 2004, Thomas et al., 2009 and Tollefsen et al., 2007) and that 0.5–1% dilutions of PW induce Vtg in juvenile cod ( Meier et al., 2010 and Sundt et al., 2011).

This threshold value was selected so as to best capture the variability of drainage densities among

the studied catchments. Four variables representing mean drainage directions were calculated, namely South, Southwest, West and Northwest. A value of 1 (or 0) means that the catchment is draining toward the named direction (or opposite to the named direction). The geographic coordinates of the flow gauging stations (latitude and longitude) were selected as two additional candidate explanatory variables (Table 2). Two soil characteristics, likely to control Venetoclax hydrological processes, were selected from the MRC soil database (MRC, 2011): soil depth and top soil texture. A four-unit scale suggested by MRC was used for

quantification (Table 1). Averaged values for each soil characteristics and each catchment were averaged by weighting each scale unit by the respective area covered in the catchment. Three land-cover types, likely to alter hydrology, were selected as candidate explanatory variables: forest, bunded rainfed lowland rice paddy fields, the majority of which is never irrigated, and wetlands, including marsh and swamp. The percentage of surface area covered by each land-cover type in each catchment was computed using the digitized 2003 land cover map of the Lower Mekong Basin prepared by MRC (2011). Forest cover was produced by merging four forest types available as separate land-cover classes in the published map: “coniferous forest”, “deciduous forest”, “evergreen forest” and “forest plantation”. The two other land-cover types were directly available since they TSA HDAC molecular weight correspond to distinct land cover classes on the published map. Table 3 presents the results of the multiple regression analyses for the 14 flow metrics listed in column 1. Column 2 provides the value of the intercept term β0. Columns 3–11 provide the coefficients βt associated with each explanatory variable Xi included in the power-law models (cf. Eq. (1)). Units of the explanatory variables are indicated in Table 2. Values of the explanatory variable “Padd” and of the flow metrics 0.50, 0.60, 0.70, 0.80, 0.90,

0.95 and Min ( Table 3) should be incremented by 1 for inclusion in Eq. (1) (cf. Section 2). As examples, Eqs. (6) and (7) show how to predict the 0.95 flow percentile (Q0.95) Diflunisal and mean annual flow (Qmean) using the coefficients provided in Table 3. equation(6) Q0.95=exp−27.857×Rain2.698×Peri1.436×Elev0.966×Lati−1.291×(Padd+1)−0.285−1Q0.95=exp−27.857×Rain2.698×Peri1.436×Elev0.966×Lati−1.291×(Padd+1)−0.285−1 equation(7) Qmean=exp−18.989×Rain2.543×Area0.883×Drai1.089Qmean=exp−18.989×Rain2.543×Area0.883×Drai1.089 In order to make the power-law models usable by a broad range of users, Table 3 presents, for each of the 14 flow metrics, an equation including climatic, geomorphologic and/or geographic explanatory variables only, exclusive of other catchment characteristics.

Although this could substantially reduce the quantity of protein required for the successful generation of TCR/pMHC complex crystals capable of diffracting to high resolution, our analyses revealed that a limited screen could exclude some important crystallization conditions for some proteins. check details Thus, our TOPS screen remains optimal for the crystallization of TCR/pMHC complexes. In conclusion, we hope that TOPS will greatly contribute to a better understanding of molecular basis for T cell recognition of self, foreign (microbial/viral/parasitic) and autoimmune antigens

by providing an improved method for generating TCR/pMHC complex protein crystals capable of high quality X-ray diffraction. Furthermore, we expect that TOPS will be useful for the determination of TCR structures in complex with classical and non-classical MHC ligands that are less well characterized, including: pMHC class II, MR1, CD1c and HLA-E. Structural information, detailing the precise atomic contacts that mediate T cell immunity, can provide clear insights into various immune dysfunctions

and could accelerate the rational design of T cell based therapies and vaccines. D.K.C., C.J.H., P.J.R., A.J.A.S., A.F., A.M.B and F.M., Belnacasan performed experiments, analyzed data and critiqued the manuscript. D.K.C., and P.J.R., conceived and directed the project. F.M., A.M.B., D.K.C., A.K.S., and P.J.R., wrote the manuscript. The authors declare no competing financial interests. No animals were used in this study. All human samples were used in accordance with UK guidelines. We thank the staff at Diamond Light Source for providing facilities

and support. FM is funded by a Tenovus PhD studentship. DKC is a Wellcome Trust Research Career Development Fellow (WT095767). PJR was supported by a RCUK Interleukin-3 receptor Fellowship. “
“Collectin 11 (CL-11), also known as collectin kidney 1 (CL-K1), belongs to the collectin group of the innate immune molecules structurally characterized by containing a carbohydrate recognition domain and a collagen-like region (Keshi et al., 2006). CL-11 is ubiquitously expressed, but highest levels are found in the adrenal glands, the kidneys, and the liver, and it is also present in circulation (Hansen et al., 2010). It is highly conserved among species ranging from zebrafish to humans. CL-11 has been shown to bind to intact bacteria, fungi and influenza A virus, and also to decrease influenza A infectivity. CL-11 was found to be associated with mannose-binding lectin-associated serine protease 1 (MASP-1) and/or MASP-3 in plasma (Hansen et al., 2010). These findings indicate a role for CL-11 in the defense against pathogens and in the activation of the complement system. Recently, CL-11 and MASP-3 were shown to be involved in fundamental developmental processes.

Briefly, the double strand DNA content was measured using Bcl-2 inhibitor a H33258 reagent after cell lysis as described by Rage et al. [23][23]. Reactive oxygen species (ROS) were measured by oxidation of dihydrodichlorofluorescein to dichlorofluorescein as described in literature [24]. Mitochondria membrane permeability (MMP) was measured by the uptake and retention of rhodamine 123 as described by Rat et al. [25][25]. ATP content was measured using the assay kit as described by the assay manual. A FCM (Becton, Dickinson and Company, New Jersey, USA) was employed to examine

the mitochondria membrane potential, cell cycle and apoptosis of HepG2 cell after treatment with AFB1 and ST. The mitochondria membrane potential (△Ψm) is measured using the JC-1 dye as described by literature report [26] by differentiation of the energized and deenergized mitochondria based on the fluorescent color. The cell cycle analysis is based the propidium selleck kinase inhibitor iodide (PI) dye that can bind double strand DNA [27], and the analysis protocol detailed in literature [28] was followed. The cell apoptosis was analyzed by employing staining reagent of PI and Annexin V-FITC as described by Vermes et al. [29][29]. In order to analyze the proapoptotic activity of AFB1 and ST in HepG2 cells, the apoptotic signaling pathway was also analyzed by immunocytochemistry

using apoptosis related markers of Bax, Bcl-2, p53, and Caspase-3. HepG2 cells at the logarithmic phase were collected at a density of 1 × 104 cells/mL. Sterile coverslips were added to a 24-well culture plate and then cell suspension was added to allow the cells seeded on the slips. After the cells become adherent, medium containing AFB1, ST and their mixture was added (in triplicate). After 48 h incubation, the slips were removed and fixed in formalin for 20 min, and then air-dried at room temperature. The slips were then hydrated through a gradient of ethanol (2 times, 1 min) -95% ethanol (2 times, 1 min) -70%

ethanol (1 time, 1 min)-water washed out after 5 min. Endogenous Ponatinib cost peroxidase activity was blocked by adding100 μL hydrogen peroxide and incubating at room temperature for 15 min, then it was washed 3 times with PBS with 5 min interval. The fixed slips were then placed in a boiling antigen retrieval solution for 15 min, incubated for 15 min, cooled out after the power is turned off and washed 3 times (5 min interval) with PBS. Non-immune serum (100 μL) from the same source of secondary antibody was added on each slip, incubated at 37 °C for 20 min, then diluted serum (antibody) (50 μL) was added (Bax 1:300, Bcl-2 1:250, Caspase3 1:200, p53 1:200) and incubated overnight at 4 °C. After incubation for 1 hr at room temperature, the slips were washed with PBS 3 times (each time 5 min). The labeled secondary antibody (50 μL) was added per slip, incubated at 37 °C for 30 min, and washed with PBS three times (each time 5 min).

ferrybox.org/euprojectferrybox/). At present, the ship-of-opportunity system is being implemented world-wide as a coastal module of the Global Ocean Observing System (GOOS, 2005 and Petersen et al., 2006). Increased interest in such unmanned systems led to the development of another component of the Europe-wide network of Ferry Box routes – the line between Gdynia (Poland) and Karlskrona (Sweden) was established at the end of 2007. Ferry Box systems improve observational capacities as they provide detailed, regular and unique data with a high temporal and spatial resolution, which

cannot be obtained on traditional oceanographic expeditions or even on regular monitoring cruises. http://www.selleckchem.com/products/Metformin-hydrochloride(Glucophage).html Obtained in a very cost-effective way, the vast amount of data supplied by Ferry Box systems can be used for validating and calibrating models; they can also be related to observations provided by satellites or aircraft (remote sensing) to reveal the spatial scales of various phenomena, thereby enabling the better resolution and understanding of marine processes (Pulliainen et al., 2003 and Ponsar et al., 2006). In the Baltic Sea, seriously affected by eutrophication (HELCOM 2009), some locations suffer from frequent cyanobacterial blooms of potentially toxic species (Wasmund, 2002 and Wasmund and Uhlig, 2003). The cyanobacteria form extensive summer

blooms and are potentially toxic towards Dasatinib manufacturer biota and human beings; they may also have adverse effects on fisheries and the recreational use of coastal areas. In order to discover the factors triggering these blooms and the environmental

consequences of the latter, the dynamics of phytoplankton HSP90 have to be studied with an appropriate spatial and temporal resolution. This paper presents an outline and preliminary results of a project, developed to set up an operational system of surveillance and registration of episodic events (e.g. harmful algal blooms) in the Baltic Sea by combining in situ measurements from a Ferry Box with satellite information. The project consisted of 3 major modules: Ferry Box, phytoplankton and satellite. The main element of this module was an autonomous ‘Ferry Box’1 system, installed on a commercial passenger ferry plying daily between Gdynia (Poland) and Karlskrona (Sweden), a distance of ca 315 km across the middle of the Baltic Proper (Figure 1). The system initially operated (2006–2008) on board m/f ‘Stena Nordica’ but was transferred to m/f ‘Stena Baltica’ in early 2009. This module provided flow-through measurements of temperature, conductivity [salinity], oxygen (oxygen results are not discussed here) and chlorophyll a fluorescence ( Table 1). The water intake for flow-through measurements and discrete sample collection was situated at ca 2 m depth.

4 and 5 It is described that pro-inflammatory cytokines, chemokines and adhesion molecules, regulate the sequential recruitment of leukocytes and are frequently observed in the tumour microenvironment6 which stimulate the growth and survival of malignant cells.7 Although the role of cytokines in tumour biology has been extensively studied, the literature is still controversial about their effects on cancer biology.8 The mediators and cellular effectors of inflammation are important components of the local tumour environment. In some types of cancer, inflammatory conditions are present before a malignant

Fluorouracil ic50 change occurs, whilst in other types of cancer, an oncogenic change induces an inflammatory microenvironment that promotes the development of tumors.9 The mechanisms of cytokines action in carcinogenesis are of great importance, due

to their involvement in tumour survival. Thus, the inhibition of pro-tumorigenic cytokine may offer an alternative target aimed at the blockage of tumour progression.10 Interleukins (IL)-4, IL-6 and IL-10 PDGFR inhibitor are multifunctional cytokines involved in adaptative and innate immunity cell mediators. The IL-10 is an immunosuppressive molecule secreted by tumours with anti-inflammatory action.11 The role of IL-10 production within the tumour microenvironment still remains controversial. It is debated that IL-10 can favour tumour growth in vitro by stimulating cell proliferation and inhibiting cell apoptosis, 1 which is correlated with poor survival of some cancer patients. 12 and 13 On the other hand, the IL-6 is a pro-inflammatory cytokine which modulates both the innate and adaptative immune response. 14 IL-6 has been shown to function as a growth factor

in several human tumors 15, 16, 17 and 18 and plays an important role in regulating apoptosis in many cell types. Interestingly, it has been demonstrated that oral squamous cell carcinoma (OSCC) patients produce increased release of IL-6 into Thiamet G saliva and that IL-6 contributes to carcinogenesis of oral mucosa or maintenance of the condition in OSCC. 19 Also, it is suggested that IL-6 inactivates p53 tumour suppressor gene. 20 In addition, IL-4 is a tumour-promoting molecule which regulates local immune response, usually elevated in human cancer patients. 21 Thus, the purpose of this study was to determine the expression of IL-4, IL6 and, IL-10 in an in vitro model of tumorigenesis, 22 which mimics a situation where in situ neoplastic cells of oral carcinoma, are surrounded by benign myoepithelial cells from pleomorphic adenoma in order to correlate the cancer cell growth and the role of these cytokines in regulating the neoplastic process.

The second dose of nimodipine was administered 24 h after the first dose in order to improve the results found in the work of Emerick et al. (2010). These strategies and the mechanisms involved beta-catenin inhibitor in the treatments of OPIDN were reviewed in a recent work published by our group (Emerick et al., 2012c). Finally, these endpoints used in the present study with methamidophos isoforms could be used to verify the neurotoxicity of other OPs that have chiral center. Most of these compounds are commercially available in

the form of the racemate, but the toxicity is enantioselective. The results presented in this study allow the identification of differences in neurotoxicities of methamidophos enantiomers and the (+)-methamidophos as the enantiomer responsible for the delayed effects. In addition, the treatments with 2 doses of nimodipine and 1 dose of Ca-glu (30 min after the first dose of nimodipine) showed to be effective to prevent the onset of OPIDN signs and lesions caused by TOCP and (+)-methamidophos. There are no conflicts of interest. Financial support for this study was

provided by the “Fundação de Amparo à Pesquisa do Estado de São Paulo” – FAPESP Grant # 2009/51048-8. Additional funding was provided by Virginia-Maryland Regional College of Veterinary Medicine. Technical assistance was provided by Elisabete Zocal P. Lepera, Luiz Potenza, Maria Aparecida dos Santos. We are also grateful to Antonio Netto Júnior for his work with the photos. “
“Carbon nanotubes (CNTs) are fiber-shaped substances that consist

of graphite hexagonal-mesh planes (graphene sheet) present as a single-layer or as multi-layers Etoposide molecular weight with nest accumulation. Tubes with single-wall structures and multi-wall structures are called single-wall carbon nanotubes (SWCNTs) and multi-wall carbon nanotubes (MWCNTs), respectively. CNTs are regarded as nanomaterials because their diameters are within the nanoscale range Epothilone B (EPO906, Patupilone) (1–100 nm). Currently, various applied studies are focusing on CNTs because of their excellent physical–chemical properties. However, there is a growing concern regarding the hazards of CNTs. Many pulmonary toxicity studies (e.g., inhalation exposure studies, intratracheal instillation studies, and pharyngeal aspiration studies) have reported that multifocal granulomas or fibrotic responses were persistently observed in the lungs of rats and mice after SWCNT exposure (Warheit et al., 2004, Lam et al., 2004, Mangum et al., 2006, Chou et al., 2008, Miyawaki et al., 2008, Shvedova et al., 2005, Shvedova et al., 2007, Shvedova et al., 2008a and Shvedova et al., 2008b). MWCNT pulmonary toxicity studies also reported similar pulmonary responses as SWCNT exposure. Granulomatous inflammation and fibrotic responses were reported in MWCNT inhalation exposure studies (Muller et al., 2005, Li et al., 2007, Ma-Hock et al., 2009 and Pauluhn, 2010).

05). Sperm samples frozen in TL-HEPES at 10 °C/min cooling rate resulted in the lowest motility (3.7%; p < 0.05). The cooling

rate significantly affected sperm motility recovery in TL-HEPES, m-KRB and TES-R treatment groups (p < 0.05). Sperm motility was significantly decreased in 10 °C/min cooling rate compared to 100 °C/min cooling rate and sperm motility increased as cooling rate increased. Membrane integrity, acrosomal integrity and MMP of frozen-thawed SD rat sperm are shown in Table 4, Table 5 and Table 6, respectively. Post-thaw membrane integrity ranged between 7.5% and 22.3% (p < 0.05). The SM, TES-R and TES-S extenders were superior for maintaining membrane integrity in sperm frozen (p < 0.05). Sperm acrosome integrity was not different among the extenders and cooling rates (p > 0.05). However, the cryopreservation caused disruption in MMP compared to fresh sperm (p < 0.05) in SD rat sperm. Motility of diluted, equilibrated Bafetinib in vitro and frozen-thawed F344 rat sperm for different extenders and cooling rates are given in Table 7, Table 8 and Table 9. Sperm motility after dilution ranged between 58.3% and 75.8% for the extenders tested. After equilibration, sperm motility loss was under 10% for all extenders. Freezing and thawing processes resulted in 27.5%

for TES-S extender at 100 °C/min cooling rate and 54.2% for TRIS-R extender at 10 °C/min cooling rate loss this website in total motility. The highest sperm motility was observed in TES-R extender (33.3%) while the lowest motility was detected in TL-HEPES extender (3.2%) at 10 °C/min cooling rate (p < 0.05). The cooling rate significantly affected

motility recovery (p < 0.05) and the highest motility was achieved in sperm exposed to TES-R and TES-S extenders at 70 and 100 °C/min cooling rates. Lower cooling rates were highly detrimental to motility (p < 0.05). Membrane and acrosome integrity and MMP of frozen-thawed F344 rat sperm for different extenders and cooling rates are given in Table 10, Table 11 and Table 12, respectively. Membrane integrity Aurora Kinase after freezing and thawing processes were between 8.8% (for TRIS-S, at 100 °C/min cooling rate) and 21.3% (for TES-S, at 70 °C/min cooling rate; p < 0.05). Post-thaw membrane integrity was lower than motility except for TL-HEPES. Sperm acrosome integrity was not affected significantly from the extenders or cooling rates (p > 0.05). But cryopreservation procedure caused the greatest disruption in MMP (p < 0.05) in F344 rat sperm. The sperm that was frozen in TES supplemented with EY, Equex Paste and sucrose or raffinose retained highest motility (p < 0.05). The strain differences in sperm motility, membrane integrity, acrosome integrity and MMP were not detected between SD and F344. In general, damage to sperm during cryopreservation have been attributed to several factors including cold shock, freezing injury, oxidative stress, alterations in membrane compositions, chemical toxicity of CPA, and osmotic stress [9].

We asked how mucoperiosteal denudation could have Ganetespib cost such a profound effect on facial growth. We first created a mouse model of mucoperiosteal denudation that specifically involved the midpalatal suture complex then used histology, immunohistochemistry, finite element (FE) modeling, micro-CT analyses, and quantitative

molecular readouts to draw a direct link between the surgical procedure, the healing response, and the resulting palatal growth inhibition. In doing so we gained critical insights into how a commonly employed surgical procedure could have the unintended consequence of impeding midfacial development. All procedures were approved by the Stanford Committee on Animal Research. Gas anesthesia was delivered to post-natal day 7 (P7) C57BL/6 pups, and the palatal mucoperiosteal denudation was performed before awakening. With the use of a dissecting microscope, a 1 mm diameter full thickness punch was made in the middle of the hard palate and the mucoperiosteum was removed with forceps; care was taken to leave the underlying skeletal tissues intact. The anterior border of the punch is made immediately posterior to the first pair of discontinuous palatal rugae (Supplemental Fig. 1B). The wound healed

by secondary intention. Age-matched littermates that were unoperated served as controls. Tissue samples were fixed in 4% paraformaldehyde at 4 °C overnight, decalcified in 19% EDTA at room temperature for 10 days, and dehydrated for paraffin embedding. Coronal sections were cut at a thickness of 8 μm. Histology was performed using Gomori Trichrome, Movat’s Pentachrome, selleck chemical and Safranin O/Fast Amino acid Green/Hematoxylin staining following standard

staining procedures [28]. Picrosirius red staining was completed and imaged under polarized light as described [28]. For alkaline phosphatase (ALP) staining, slides were pre-incubated in NTMT buffer for 15 min and then stained in ALP solution containing 2 mL NTMT, 10 μl NBT (Roche), and 7.5 μl BCIP (Roche) for 30 min at 37 °C. Tartrate resistant acid phosphatase (TRAP) staining was performed using a Leukocyte Acid Phosphatase Kit (Sigma, St. Louis, MO). Immunohistochemistry for Ki67, Osteopontin, collagen I, collagen II, and X was carried out as described [28]. In brief, slides were immersed in 0.2% Triton for 5 min then incubated in Antigen Unmasking Solution (Vector Laboratories, diluted 1:100) at 95 °C for 20 min. After returning to room temperature slides were immersed in 3% hydrogen peroxide for 5 min and blocked in 5% goat serum for 30 min. Slides were incubated in corresponding primary antibodies (Ki67 rabbit polyclonal antibody, Thermo Scientific, diluted 1:100; Osteopontin rabbit polyclonal, Abcam, diluted 1:2000; Collagen I rabbit polyclonal antibody, Calbiochem, diluted 1:500; Collagen II rabbit polyclonal antibody, Millipore, diluted 1:50; Collagen X rabbit polyclonal antibody, Calbiochem, diluted 1:500) overnight at 4 °C.