[9] Of note, his illustration also clearly demonstrates a sharp, oblique boundary between lesioned CA1 sector and well-preserved subiculum, which represents the subicular-CA1 border zone or “prosubiculum” of Lorente de Nó.[8] In fact, his description represents the most common and characteristic histological feature of HS. In 1966, Margerison and Corsellis defined two types of hippocampal damage.[10] One was a pattern previously characterized by Bratz’ description and termed “classical” Ammon’s horn sclerosis. Pirfenidone Another pattern of hippocampal damage that they described was characterized by neuronal loss confined

to the hilus of the dentate gyrus or “end folium”, termed “end folium sclerosis (EFS)”. In addition to these two patterns of HS, Bruton added, in his monograph published in 1988, a third pattern of HS called “total” Ammon’s horn sclerosis, showing almost complete neuronal loss in all sectors of the hippocampus.[11] These specific patterns of HS could easily

be assessed based solely on qualitative observation; however, Bruton found no apparent correlation between any of these specific types of HS and the clinical history among 107 patients in his study. HDAC inhibitor Since then, several proposals for classification and a grading system for HS have been published (Table 1). The first systematic attempt to semi-quantitatively evaluate the severity of hippocampal neuronal loss for the histological grading of HS was proposed by Wyler et al. in 1992, providing four grades for HS along with a diagnosis of no HS introducing the term “mesial temporal damage (MTD)”.[12] Wyler’s grading system revealed that classical and total Ammon’s horn sclerosis were the most frequent pathologies in mTLE. Inverse clinicopathological correlation has been reported between Wyler’s grade and postsurgical memory impairment; patients having the most postoperative memory loss were the ones with normal or grade I pathology,

whereas those patients with high-grade (III and IV) pathologies Nintedanib (BIBF 1120) showed little in terms of significant postoperative memory problems.[15] Mossy fiber sprouting in the dentate gyrus as demonstrated by Timm’s staining can be observed in cases with Wyler’s high-grade lesions.[16] In terms of memory impairment, histological patterns of granule cell pathology in the dentate gyrus have been reported to be associated with learning dysfunction in addition to older age at epilepsy surgery and longer duration of illness.[17] A more recent study has demonstrated that the in vitro capacity of proliferation and differentiation into neurons of neural stem cells isolated from the dentate gyrus in patients with pharmacoresistant mTLE was significantly associated with preoperative memory performance and the number of granule cells in the resected specimen.

Cells were incubated at 37 °C in 5% CO2. On the day of tumour challenge, TC-1 cells Fludarabine molecular weight were harvested by trypsinization, washed with

phosphate-buffered saline (PBS), counted and finally resuspended in 500 μl of PBS. Plasmid DNA construction.  The generation of pcDNA-E7 (E7 Genebank accession number K02718, 294 bp, kindly provided by Prof. T.C. Wu, John Hopkins Medical Institutions, USA) and pQE-(NT-gp96) has been described previously [27]. For construction of pUC-E7, the E7 fragment was first amplified with PCR using pcDNA-E7 as the template and a set of primers designed as follows: E7F: 5′-GGGGATCCACCATGCATGGAGATACACCT-3 E7R: 5′-ATAAGCTTCCCGGGTGGTTTCTGAGAACA-3 The BamHI restriction site in forward primer and HindIII and SmaI restriction sites in reverse primer were underlined. PCRs were performed under conditions including 95 °C, 30 s; 67 °C, 30 s; 72 °C, 1 min for a total of 30 cycles. The amplified

product was then cloned into the BamHI/SmaI sites of the pUC18 cloning vector (Fermentas). To prepare plasmid DNA pDrive-(NT-gp96) (gp96 gene was kindly provided by Dr. Jacques Robert, University of Rochester Medical Center, USA), PCR was performed using pQE-(NT-gp96) as template and a set of primers (The SmaI in forward primer and KpnI restriction sites in reverse primer were indicated in bold): NTgp96FF: 5′-CGGCCCGGGGAAGATGACGTTGAA-3 gp96RN: 5′-ATGAGCTCGGTACCTTTGTAGAAGGCTTTGTA-3 The amplification program for performing PCR was as follows: 95 °C, 1 min; 62 °C, 2 min; 72 °C for 1.5 min for MEK inhibitor a total of 30 cycles. The PCR product

was cloned in pDrive cloning vector according to kit instruction (Qiagen® PCR cloning kit, Hilden, Sodium butyrate Germany). As the PCR product could insert in both direct and reverse orientation, therefore the direct-oriented clone was selected using PstI endonuclease which cut the NT-gp96 gene and also exist in multiple cloning site of pDrive. The PstI digestion resulted in 905 and 2945 bp fragments in direct-oriented pDrive-(NT-gp96) clone. To generate pUC-(E7-NT-gp96), the NT-gp96 fragment was isolated from pDrive-(NT-gp96) and then cloned into the SmaI/SacI sites of pUC-E7. DNA sequencing was performed to confirm the pUC-(E7-NT-gp96). For protein expression, the E7-NT-gp96 gene was digested from pUC-(E7-NT-gp96) and then cloned in BamHI/SacI sites of pQE-30 expression vector (Qiagen, Germany). Expression and purification of the recombinant E7-NT-gp96 [rE7-NT-gp96].  The production and purification of rE7 and rNT-gp96 were carried out as previously described [27]. E. coli strain M15 transformed with the recombinant pQE-(E7-NT-gp96) was grown at 37 °C in LB medium supplemented with 100 μg/ml ampicillin and 25 μg/ml kanamycin (Sigma, Germany).

“
“Please cite this paper as: Wagner R, Modla S, Hossler F, Czymmek K. Three-dimensional analysis and computer modeling of the capillary endothelial vesicular system with electron tomography. Microcirculation 19: 477–484, 2012. Objective:  We examined the three-dimensional organization of the endothelial vesicular system with TEM tomography of semi-thick sections. Materials and methods:  Mouse abdominal muscle capillaries were perfused with terbium to

label vesicular compartments open to the luminal surface. The tissue was prepared for TEM and semi-thick (250 nm) sections were cut. Dual axis tilt series, collected from +60° to −60° at 1° increments, were acquired Selleckchem Romidepsin in regions of labeled abluminal caveolae. These tomograms were reconstructed and analyzed to reveal three-dimensional vesicular

associations not evident in thin sections. Results:  Reconstructed tomograms revealed free vesicles, both labeled and unlabeled, in the endothelial GS-1101 cost cytoplasm as well as transendothelial channels that spanned the luminal and abluminal membranes. A large membranous compartment connecting the luminal and abluminal surfaces was also present. Computer modeling of tomographic data and video animations provided three-dimensional perspectives to these structures. Conclusions:  Uncertainties associated with other three-dimensional methods to study the capillary wall Tyrosine-protein kinase BLK are remedied by tomographic analysis of semi-thick sections. Transendothelial channels of fused vesicles and free cytoplasmic vesicles give credence to their role as large pores in the transport of solutes across the walls of continuous capillaries. Membranous vesicular compartments are the most conspicuous features in the cytoplasm of capillary endothelial cells. Although there is substantial evidence that they are

involved in transendothelial transport, the mechanism(s) of this transport and its structural correlates are unclear. Ultrastructural analysis of the capillary endothelial system has been impaired by the lack of three-dimensionality of conventional TEM sections. Stereoviewing of tilted thick sections with high-voltage EM [26] is limited to a single viewing angle and structures are often obscured by the overlapping of details in two-dimensional images recorded from thicker sections. Reconstruction of serial ultrathin sections [1,4,5] requires difficult to obtain ribbons of ultrathin sections and are constrained to small sampled regions. Moreover, serial sections are limited in the z-resolution to twice the section thickness, making nanometer-sized details difficult to resolve.

Two hundred and twenty-five patients have been recruited within a collaborative project (GenHomme, Research French ministry) involving the Nantes Institute of Transplantation,

the Center for Adult Transplantation of the Necker Hospital (Paris, France) and the Biotechnology Company, TcLand Expression (Nantes, France). Sixty-one additional patients were recruited in the framework of the European “Indices of Tolerance” Network. Selleck Tipifarnib The protocol of the study was approved by the Ethical Committees of Nantes and Paris Universities and of the European Commission. All patients signed a written informed consent before inclusion. Several different clinical groups were studied (Table 1). Operationally tolerant patients (TOL, n=14) are defined by a stable kidney graft function (Creatininemia<150 μmol/L, Proteinuria<1 g 24 h−1) off immunosuppressive drugs for more than 1 year (mean drug-free duration=8.3±5.7 years) at the time of testing. This definition fulfills Cabozantinib EU criteria for operational tolerance (for review, see 4). Immunosuppressive treatment, including corticosteroids, was

stopped on account of non-compliance (n=11), calcineurin inhibitor toxicity (n=1), post-transplant lymphoproliferative disorder (n=1) or cancer (n=1). Patients with the “suspicious” form of chronic humoral rejection (CHR, n=21) all had a progressive degradation of their renal function (Creatininemia >150 μmol/L and Proteinuria >1 g 24 h−1). In all cases, transplant renal biopsies documented histological signs of chronic humoral rejection at the time of the blood test (Banff 05 grade II or IIIb) with either C4d deposition (in 14 patients out of 21) or circulating anti-donor class II Ab in 11 out of 21 patients. Because the patients had not necessarily both circulating anti-donor class II Ab and

Olopatadine C4d deposits, they were referred to as “suspicious” of chronic humoral rejection, as suggested by Banff ’07 classification 2. Long-term stable patients (n=229) comprised patients who had stable kidney graft function on immunosuppresants (either mycophenolate mofetil or azathioprine), supplemented with calcineurin inhibitors treatment in some (n=209 referred as STA) but not in others cases (n=8, referred as STN). Patients also received corticosteroids. The cohort of 209 STA patients is composed of 182 patients recruited from the GenHomme study (patients who have been transplanted at least 5 years previously) and 27 patients from the “Indices of Tolerance” network. Patients were included based on the function of their kidney graft assessed at least 5 years after transplantation (Creatininemia <150 μmol/L, Proteinuria <1 g 24 h−1). Ongoing infection and episodes of rejection defined the exclusion criteria.

Incidentally, Fujii et al. 8 reported a phenomenon describing NKT cell turnover, a decrease in the NKT cell population on day 1 after α-GalCer administration later found to be due to TCR down-regulation, after administration of free α-GalCer that was “less rapid and severe” when DCs pulsed with α-GalCer were administered. Antigen-specific cellular LDK378 datasheet immune responses were measured after each dose of the α-GalCer adjuvant and OVA antigen mixture, similar to our previously reported studies with a different antigen 7. Both these studies demonstrate that multiple doses of α-GalCer, administered by the intranasal route, are necessary to induce

efficient antigen-specific cellular immune responses, regardless of the mouse strain used. In addition to the antigen-specific cellular immune responses, effectiveness of α-GalCer as an adjuvant after intranasal immunization to induce humoral immune responses, in terms of antigen-specific IgA and IgG responses has been described in the literature

24 and also observed in other unrelated studies in our laboratory (data not shown). Thus, our studies provide mechanistic support for mucosal delivery of α-GalCer adjuvant as an attractive strategy for vaccination regimens. It is also important to note potential inflammatory effects from the intranasal administration of α-GalCer. Different mouse model studies revealed that intranasal administration of α-GalCer can induce airway infiltration of a combination of eosiniphils, neutrophils, Selumetinib concentration and/or monocytes 25, 26. Preliminary studies in our lab showed increase in the percentages of eosinophils but not neutrophils or monocytes (data not shown). However, clinical trials performed by Kunii et al. 4 showed that administration of α-GalCer by a nasal sub-mucosal route was safe. Overall, this investigation has shown that α-GalCer can be administered by the intranasal route for primary and booster immunizations to induce cellular immune responses to co-administered antigens, without inducing NKT cell anergy. This is in striking contrast to α-GalCer administration

by the intravenous route, in which a single dose leads to NKT cell anergy and a reduction in the ability of the adjuvant to boost adaptive immune responses to co-administered antigen. Thus, selleckchem our data support the intranasal route of immunization as an attractive route for immunization especially because the ability to deliver multiple doses of the vaccine is essential for most therapeutic applications against infectious diseases and cancer. Female C57Bl/6 mice aged 6–10 wk were purchased from the National Cancer Institute. All procedures on the animals were carried out in accordance with institutionally approved protocols. The animals were housed in microisolator cages and provided with sterile food and water. The animal facility is fully accredited by the Association for Assessment and Accreditation of Laboratory Animals Care International.

There were no operative complications, no flap-related complications, and at two years follow-up, the patient subjectively described bilateral soft and supple breasts, which were symmetrical in a bra, and with which she has reported high satisfaction. An account of the “split DIEP flap” is provided, highlighting the planning, technique, check details and vascular rationale. The technique comprises partition of a previously transferred DIEP flap breast reconstruction into two parts based on preoperative computed tomographic angiography, performed to guide surgical planning in avoiding pedicle

damage and identifying the portion of the flap to island. The split DIEP flap for staged bilateral autologous breast reconstruction offers two soft-tissue flaps for the price of one donor site, offering new possibilities in breast reconstruction and the broader field of tissue transplantation. © 2013 Wiley Periodicals, Inc. Microsurgery, 2013.

“
“Reconstruction of distal thumb injuries still remains a challenge for hand surgeons. Surgical treatment includes the use of local, regional, and free flaps. The purpose of this report is to present the results of the use of a sensitive reverse flow first dorsal metacarpal artery (FDMA) flap. The skin flap was designed on the radial side of the proximal phalanx of the index finger based on the ulnar and radial branch of the FDMA and a sensory branch of the superficial radial nerve. This neurovascular flap was used Selleckchem PF 2341066 in five patients to cover distal soft-tissue thumb

defects. All flaps achieved primary healing except for one patient in whom superficial partial necrosis of the flap occurred, and the defect healed by second intention. All patients maintained the thumb original length and were able to return to their previous daily activities. The reverse flow FDMA flap is a reliable option to cover immediate and delayed defects of distal thumb, offering acceptable functional and cosmetic outcomes in respect to sensibility, durability, and skin-match. © 2013 Wiley Periodicals, Inc. Microsurgery 34:283–286, 2014. “
“To investigate the relationship between ischemic time and rejection against allotransplants, vascularized cutaneous flaps from the groin http://www.selleck.co.jp/products/azd9291.html of Brown Norway rats were transplanted to Lewis rats. The ischemic time was set at 1 hour and 6 hours for comparison. Cycrosporine A was used as the immunosuppressant. The results showed more severe rejection in the 6 hours ischemic time group in vivo, and in vitro examination using mixed lymphocyte reaction assay also demonstrated a greater antidonor response in 6 hours-ischemic group than that in 1 hour-group. Immunohistochemical study demonstrated more MHC class II antigen expression in 6 hours-ischemic group than in 1 hour-group. These results suggest that longer ischemic time induces more severe rejection against allo-transplanted tissue compared with the shorter one through an upregulation of MHC class II antigen.

The finding selleck inhibitor that there are cross-reactive epitopes

in the NCRD of SP-D and bovine collectins will be useful in efforts to identify binding sites of these functionally enhancing mAb. Future studies will involve development of other combined mutants (e.g., with substitutions of D325 and R343) in efforts to specifically increase antiviral activity further. This work was supported by NIH Grant AI-83222 (KLH, ECC and JH) and Grant HL069031 (KLH). “
“Germinal centre (GC) reactions are central features of T-cell-driven B-cell responses, and the site where antibody-producing cells and memory B cells are generated. Within GCs, a range of complex cellular and molecular events occur which are critical for the generation of learn more high affinity antibodies. These processes require exquisite regulation not only to ensure the production of desired antibodies, but to minimize unwanted autoreactive or low affinity antibodies. To assess whether T regulatory (Treg) cells participate in the control of GC responses, immunized mice were treated with an anti-glucocorticoid-induced tumour necrosis factor receptor-related protein (GITR)

monoclonal antibody (mAb) to disrupt Treg-cell activity. In anti-GITR-treated mice, the GC B-cell pool was significantly larger compared with control-treated animals, with switched GC B cells composing an abnormally high proportion of the response. Dysregulated GCs were also observed regardless of strain, T helper type 1 or 2 polarizing antigens,

and were also seen after anti-CD25 mAb Neratinib ic50 treatment. Within the spleens of immunized mice, CXCR5+ and CCR7− Treg cells were documented by flow cytometry and Foxp3+ cells were found within GCs using immunohistology. Final studies demonstrated administration of either anti-transforming growth factor-β or anti-interleukin-10 receptor blocking mAb to likewise result in dysregulated GCs, suggesting that generation of inducible Treg cells is important in controlling the GC response. Taken together, these findings indicate that Treg cells contribute to the overall size and quality of the humoral response by controlling homeostasis within GCs. The central feature of primary T-cell-driven B-cell responses is the germinal centre (GC) reaction. The GCs are structures that form within the follicles of secondary lymphoid organs after challenge with T-cell-dependent antigens. They consist of several key cell types, including specialized CD4+ T follicular helper (Tfh) cells, antigen-selected B cells and follicular dendritic cells.1–4 Importantly, GCs generate high-affinity plasma cells and memory B cells, which produce antibodies crucial for clearing the offending antigen and protecting the host upon secondary exposure.

However, during neurodegeneration function could be dramatically altered by the aggregation of phosphorylated tau

protein. Interestingly, prior to formation of NFT alterations, neurone functioning could be compromised. Here, we believed that the study of pretangle like structures could become a more suitable research model in order to find the pathogenesis of such complex tau diseases. Overall, our findings document a well-defined pattern of phosphorylation and sequential or simultaneous cleavage of tau selleck at D421 in both AD and DS, with phosphorylation at sites Ser396–404 being one of the earliest events. Finally, these data validate PHF-1 as an efficient marker for AD cytopathology following the progression of tau aggregation into NFT. We thank to Peter Davis for PHF-1 antibody donation. We thank Katarina Stojkovic for critical comments. Work in the authors’ laboratories is supported by Consejo Nacional de Ciencia y Tecnología (Conacyt), selleckchem Mexico; Canadian Institutes of Health Research (CIHR), Canada and Fonds de la recherche en santé du Québec (FRSQ), Québec, Canada. This project was supported by grants from the National Center for Research Resources

(5 G12RR013646-12), the National Institute on Minority Health and Health Disparities (G12MD007591) from the National Institutes of Health, and from the Research Centers in Minority Institutions (RCMI). S.M.-R. was awarded with a postdoctoral scholarship support FRSQ, Canada. Conceived and designed

the experiments: S.M.-R. Performed the experiments: S.M.-R. and J.L.-M. Analysed the data: S.M.-R., G.P. and M.C.A.-A. Contributed reagents/materials/analysis tools: G.P., M.C.A.-A. and S.W. Wrote the paper: S.M.-R. Financial support: G.P. and S.W. All authors read and approved the final manuscript. “
“Basophilic inclusions (BIs), which are characterized by their staining properties of being weakly ID-8 argyrophilic, reactive with Nissl staining, and immunohistochemically negative for tau and transactive response (TAR) DNA-binding protein 43 (TDP-43), have been identified in patients with juvenile-onset amyotrophic lateral sclerosis (ALS) and adult-onset atypical ALS with ophthalmoplegia, autonomic dysfunction, cerebellar ataxia, or a frontal lobe syndrome. Mutations in the fused in sarcoma gene (FUS) have been reported in cases of familial and sporadic ALS, and FUS immunoreactivity has been demonstrated in basophilic inclusion body disease (BIBD), neuronal intermediate filament inclusion disease (NIFID), and atypical frontotemporal lobar degeneration with ubiquitin-positive and tau-negative inclusions (aFTLD-U). In the present study, we immunohistochemically and ultrastructurally studied an autopsy case of sporadic adult-onset ALS with numerous BIs.

Each data was analyzed by multivariate analysis. Results: Serum levels of IgA, Gd-IgA1, Gd-IgA1-specific IgG and Gd-IgA1-specific IgA were elevated in patients with IgAN compared with disease controls and healthy controls. However, none of the biomarkers alone fully differentiated IgAN patients from disease controls. Therefore, we re-analyzed these biomarkers and compared them with clinical data, such as age, gender, serum creatinine, urinary protein/creatinine ratio and degree of microscopic hematuria by combination. In accordance of analysis, we omitted

unnecessary variables from analysis using Principal Component Analysis (PCA) that is a variable reduction procedure to analyze the importance of each variable. Then, each CH5424802 solubility dmso variable were analyzed using logistic model. This model differentiated IgAN patients from disease controls with 81% specificity and 91% sensitivity. Conclusion: Our results suggest that serum Gd-IgA1 and Gd-IgA1-specific antibodies (IgG and IgA) are useful biomarkers for diagnosis of IgAN. The novel quantitative scoring system can be applied for diagnosis of IgAN besides renal biopsy. SUZUKI HITOSHI1, YANAGAWA HIROYUKI1, SUZUKI YUSUKE1, SATAKE KENJI1, JULIAN BRUCE A2,3, NOVAK JAN3, TOMINO YASUHIKO1 1Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine; 2Department of Medicine, University of Alabama at Birmingham;

3Department of Microbiology, University of Alabama at Birmingham Introduction: IgA nephropathy (IgAN) is an autoimmune BVD-523 purchase glomerulonephritis that immune complexes (IC) composed of galactose-deficient IgA1 (Gd-IgA1) and

anti-glycan IgG autoantibodies deposit in the glomeruli. Serum levels of Gd-IgA1 as well as anti-glycan IgG autoantibodies, responsible for the MycoClean Mycoplasma Removal Kit formation of ICs with Gd-IgA1, are elevated in patients with IgAN. However, the pathogenic roles of Gd-IgA1-containing IC and mechanisms of immune deposits in the mesangium are still obscure. Methods: Polymeric Gd-IgA1 myeloma protein and recombinant anti-glycan IgG were used to form IC (Gd-IgA1-IgG IC) in vitro to inject i.v. into nude mice. After various time intervals, mice were sacrificed; blood and urine were collected to determine serum IgA1 and IgG, urinary protein and creatinine and hematuria. Furthermore, to assess the potential capacity of these IC to activate endothelial cells, human renal glomerular endothelial cells (HRGEC) were co-cultured with Gd-IgA1 alone or Gd-IgA1-IgG IC for 72 h. Then, transcript levels of TNF-a, TGF-b, IL-6, ICAM1 and E-selectin in HRGEC were measured by RealTime PCR. Results: Gd-IgA1 and anti-glycan IgG formed IC that deposited with murine C3 in the mesangium and in small amounts in the subendothelial area of the glomerular capillaries, and induced hematuria and proteinuria. In control mice injected with only Gd-IgA1 or Gd-IgA1 with IgG from a healthy control, IgA1 deposited only transiently and did not cause tissue injury.

After electrophoresis, the proteins were blotted onto a PVDF membrane according to selleck products standard protocols. After blocking in 5% non-fat milk, the membrane was incubated with the appropriate primary antibody (anti-iNOS, 1 : 500 or anti-SOCS-1 1 : 1000) overnight at 4°, and with the appropriate secondary antibody (1 : 10 000) (GE Healthcare, Waukesha, WI) for 2 hr at room temperature. Equal protein loading was shown by re-probing the membrane with an anti-actin antibody (1 : 10 000) (Sigma) and with

the appropriate secondary antibody. After this incubation period, the blots were washed several times with saline buffer (TBS/T – 25 mm Tris–HCl, 150 mm NaCl, 0·1% Tween) and incubated with ECF substrate (enhanced chemifluorescence substrate) (alkaline phosphatase substrate; 20 μl ECF/cm2 of membrane) for 5 min at room temperature and then submitted to fluorescence detection at 570 nm using a Molecular Imager Versa Doc MP 4000 System (Bio-Rad). For each membrane, the analysis of band intensity was performed using the Quantity One software (Bio-Rad). Nitric oxide production was assessed by the Griess Reagent System (Promega Corporation, Madison, WI), a colorimetric assay that detects the presence of nitrite (), a stable reaction product of nitric oxide (NO) and molecular oxygen. Briefly, 50 μl cell medium, collected from each well, was incubated

learn more for 5 min with 50 μl sulfanilamide, followed by a further incubation of 5 min with 50 μl of N-1-napthylethylenediamide. The optical density of the samples was measured at 540 nm in a microplate ID-8 reader and the nitrite concentration was determined by comparison with a standard curve obtained for a solution of sodium nitrite prepared

in RPMI-1640. Immunocytochemistry studies were performed in N9 microglia cells according to established protocols. Briefly, following transfection and LPS exposure, cells were washed twice with PBS and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. The cells were then permeabilized for 2 min with 0·2% Triton X-100 and non-specific binding epitopes were blocked by incubating the cells for 30 min with a 5% BSA solution prepared in PBS. Cells were incubated overnight at 4° with primary antibodies against the CD11b integrin (1 : 500) and α-tubulin (1 : 1000) prepared in PBS containing 1% BSA. Following two washing steps with PBS, cells were incubated for 2 hr at room temperature with the respective secondary antibodies (anti-rat Alexa Fluor-594 conjugate and anti-rabbit Alexa Fluor-488 conjugate; Molecular Probes, Leiden, the Netherlands) diluted 1 : 500 in PBS containing 1% BSA. Finally, all coverslips containing the samples were rinsed twice in PBS and incubated in the dark with DAPI (1 μg/ml) for 5 min, before being mounted on glass slides using Moviol (Sigma).