2C, top). The same results were obtained when viral titers in IgMi mice after

LCMV Docile infection were analyzed (Fig. 2C, bottom). Taken together, these data suggested that Abs induced in the early phase of an LCMV Docile GSK2126458 research buy infection were required to prevent T-cell exhaustion and viral persistence. Due to the phenotype of Ab-deficient mice after LCMV Docile infection, we used this viral strain for all subsequent experiments of this study. Next, we determined the kinetics of the LCMV-specific Ab response in B6 mice using a newly established sensitive sandwich ELISA as detailed in the Material and methods. LCMV-specific IgG titers in serum of LCMV Docile infected mice strongly increased between days 6 and 8 and reached maximal levels 2 weeks p.i. (Fig. 3A, filled circles). The IgG response was T-cell help dependent since Ab titers were strongly decreased in CD4+ T-cell-depleted mice (Fig. 3A, open circles). The viral antigen specificity of immune serum taken from LCMV Docile infected mice at d20 p.i. was analyzed by immunoprecipitation and immunoblotting. The results revealed that LCMV immune serum predominantly

contained Abs specific for LCMV NP (Fig. 3B) confirming previous data [14]. Importantly, virus neutralizing activity was never observed in these LCMV immune sera even when used at a high concentration (Fig. 3C). To provide additional evidence for the lack of virus neutralizing activity, virus serum mixes (90% www.selleckchem.com/products/idasanutlin-rg-7388.html serum) were incubated overnight before inoculation into mice. Two days after inoculation,

LCMV titers in spleens were enumerated. The neutralizing LCMV GP specific mAb KL25 was used as a positive control in these assays. As shown in Fig. 3D, treatment with mAb KL25 completed prevented infection whereas preincubation with LCMV immune serum did not affect initial viral replication. Having shown that mice with impaired humoral immunity were unable to control LCMV Docile infection, we next wondered whether transfer of LCMV immune serum could accelerate virus clearance. First, LCMV Docile infected MD4 and IgMi mice were treated Dynein with LCMV immune sera free of infectious virus that were obtained from infected wild-type mice at day 20 p.i. Viral titers in spleen, liver, and lungs were determined 14 days later. This treatment was able to lower viral titers in some mice but the antiviral effects were variable, particularly when using MD4 mice (Fig. 4). To obtain a more robust read-out for the potential antiviral activity of LCMV-specific Abs, we next tested B6 wild-type mice as hosts. Mice were infected with LCMV Docile and at day 1 serum from healthy uninfected mice (= normal serum) or LCMV immune serum was injected i.p. and the kinetics of viral elimination was followed. At day 2 and day 4 p.i., viral load between the two groups did not significantly differ (Fig. 5).

We have shown that CGRP is up-regulated in invading macrophages in injured rat sciatic nerves and, through an autocrine or paracrine mechanism it contributes Angiogenesis inhibitor to the up-regulation of the pro-inflammatory cytokine interleukin-6 (IL-6) in invading macrophages.10 In rat peritoneal

macrophages, the endotoxin lipopolysaccharide (LPS) increased CGRP contents in a concentration-dependent manner.10 These data suggest that CGRP is produced by macrophages following inflammatory stimulation and its up-regulation in macrophages affects the functions of invading macrophages, hence influencing the outcome of inflammation. Monocytes/macrophages are the main effector cells of the immune system and play an essential role in host defence mechanisms against infectious micro-organisms and tumour cells. By secreting numerous biologically active molecules,

macrophages are involved not only in the regulation of the secondary immune response, but also in the process of inflammation and tissue repair. A growing body of evidence suggests that CGRP plays an important role in regulating the functions of macrophages, including the production of inflammation-related chemokines and cytokines. It is therefore important Rapamycin to understand how CGRP is up-regulated in macrophages during the inflammatory response and which functions of macrophages are modulated by CGRP. To address these issues, we used the RAW 264.7 murine macrophage cell line to obtain a large quantity of homogeneous macrophages and LPS as a prototype of inflammatory stimulus to examine the possible factors that can induce CGRP in RAW macrophages. The first aim of the present study was to determine whether LPS could induce CGRP in the RAW macrophage cell line. Lipopolysaccharide see more has been reported to up-regulate

the expression of inflammatory mediators such as IL-1β, tumour necrosis factor-α (TNFα), IL-6, nerve growth factor (NGF), inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2) in RAW264.7 macrophages.11–13 The nuclear factor-κB (NF-κB) signalling pathway is involved in LPS-induced production of inflammatory mediators in this cell line.12,14 As a transcription factor, NF-κB plays a key role in the transcriptional regulation of genes of numerous inflammatory mediators including iNOS, COX2, IL-1β, IL-6 and TNFα.15 Among the inflammatory mediators, NGF,7,9 IL-1β,16,17 IL-618 and TNFα19 have been shown to induce CGRP in human B lymphocytes, monocytes, sensory neurons and various other cell types. Hence, the second aim of this study was to investigate the inflammatory mediators likely to be involved in LPS-induced CGRP in RAW macrophages and whether LPS-induced CGRP is mediated through the NF-κB signalling pathway.

, 2008), even in culture-negative cases. Stoodleyet al. (2008) have also published GDC 0449 confocal micrographs showing the consistent presence of biofilms of live coccoid bacterial cells (using Molecular Probes Live/Dead BacLite Kit) in an infected elbow case (Fig. 1) that yielded negative cultures over a period of 5 years,

during which the clinical state of the patient necessitated several serious replacement procedures. The confocal data were supported by positive reverse transcriptase-PCR results for bacterial mRNA for Staphylococcus aureus. The orthopedic problem that offers the most dramatic contrast between culture data and modern molecular methods of diagnosis is the tragic problem of the Sulzer acetabular cup. When a critical nitric acid washing step was INK 128 solubility dmso omitted from the manufacturing process for this device, the microbial biofilms accreted during manufacture were retained and, even though ethylene oxide sterilization killed the sessile bacteria, the residual polysaccharides of the matrix increased the colonization potential of these devices. Approximately 1500 cases of ‘aseptic loosening’ resulted, and this designation was made because the culture results were consistently negative

for both aspirates and interoperative specimens (Effenbergeret al., 2004). We have examined a subset of eight of these ‘aseptic loosenings’ and, in each case, we have found direct evidence of the presence of bacteria on explants at the time of revision. Figure 2 shows unequivocal evidence of the presence of coccoid bacterial cells on the surface of a culture-negative Sulzer acetabular cup explanted from a case of so-called ‘aseptic loosening.’ These cells were seen to form slime-enclosed biofilm microcolonies on the plastic surface. When these acetabular cups were reacted with species-specific FISH probes for Staphylococcus epidermidis, the bacterial cells showed fluorescence (Fig. 2, inset), and the cells were seen to be growing in coherent biofilms. Because the detection of bacteria like S. aureus is pivotal in many clinical decisions in orthopedic surgery, and because the presence of methicillin-resistant

S. aureus (MRSA) can pose intractable problems, it may be valuable to address the culture of the biofilm phenotype of however this organism. Extensive studies of the distribution of S. aureus in the human female reproduction tract were triggered by the threat of toxic shock, caused by the secretion of the TSST1 toxin produced by this organism; hence, we explored their detection and characterization using culture methods and new molecular techniques (Veehet al., 2003). In a survey of 3000 healthy volunteers, using very careful culture techniques in which vaginal swabs were carried to the lab at body temperature and fresh moist plates were used, positive cultures were obtained from 10.8% of these women. This percentage was slightly higher than that found in several previous studies (Wiseet al.

It is speculated by these authors that in vivo, CTLA-4 could originate from TRegs to stimulate see more IDO from decidual DCs. The protective role for CTLA-4 in pregnancy is supported by the fact that both the number of TRegs and the level of CTLA-4 on TRegs are lower in the decidua in cases of spontaneous abortion.36 CTLA-4 expression by placental fibroblasts has also been reported,37 providing another potential source of ligand to mediate reverse signaling at the maternal–fetal interface. IDO production following reverse signaling may also occur in placental macrophages. Hofbauer cells express both IDO22 and B7-2,25 suggesting that CLTA-4 ligation of B7-2 on

placental macrophages may induce IDO from these cells in the same manner as in decidual DCs.35 The role of B7-1 and B7-2 in pregnancy has been investigated in the ‘abortion-prone’ CBA × DBA mouse model using blocking antibodies administered at approximately the time of implantation. It was reported that blocking the signaling pathway of both B7-1 and B7-2

or B7-2 alone improved viability of fetuses38. This was accompanied by an increase in the percentage of CD4+ CD25+ Treg cells expressing CTLA-4 as well click here as skewing toward a Th2 response. These authors also found that unfractionated T cells transferred from anti-B7-1/-2-treated mice into subsequent CBA × DBA matings were protective, suggesting that an anti-B7 antibody-induced population of TRegs was able to suppress endogenous maternal immune reactivity to the fetus.39 However, these results were not supported in another abortion-prone

model of allogeneic pregnancy, where blocking B7-2 did not improve fetal viability in CBA × B6 breedings.40 B7-H1 was identified 10 years ago through searches of the expressed sequence tag database for molecules containing homology to B7-1 and B7-2.41,42 It shares approximately 20% amino acid sequence identity with B7-1 and B7-2. Such low levels of sequence homology are commonly seen among members of the B7 family, which instead share high levels of similarity in their secondary and tertiary structures.43 B7-H1 mRNA has been found P-type ATPase to be broadly distributed among many tissues, but its protein distribution is more restricted, suggesting that post-transcriptional mechanisms may have an important role in controlling B7-H1 expression, an idea that has received experimental support from our laboratory and others.44,45 However, its expression can also be induced in parenchymal cells of most organs under inflammatory conditions. Constitutive B7-H1 expression occurs on only a few cells: APCs, lymphocytes, cardiac endothelial cells, and, notably, trophoblast cells. The cellular expression on both APCs and parenchymal cells reflects the ability of B7-H1 to interfere with T-cell activation at both the priming and effector stages of the immune response within lymphoid organs and peripheral tissues, respectively.

Peripheral blood and colon or small intestinal specimens were obtained

from IBD patients undergoing small intestinal resection or subtotal colectomy. The rectal specimens were obtained from patients undergoing proctectomy prior to the construction of a pelvic pouch. The patients were either in remission or in a chronic phase of the disease, the former undergoing different forms of reconstructive surgery while the latter were undergoing surgery with curative intent due to active disease. The diagnosis for each patient was determined on the basis of past and present clinical parameters and histopathological criteria post-surgery. The control group consisted of healthy Vincristine clinical trial volunteers (peripheral blood) and patients undergoing therapeutic bowel resection for adenocarcinomas (peripheral blood and colonic specimens). For the specimens from the adenocarcinoma patients only tissue from uninvolved colon was used. The data on the participating individuals are shown in Table 1. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll/Hypaque (Amersham Biosciences AB, Uppsala, Sweden) Saracatinib in vivo density gradient

centrifugation. When indicated, cells were stained with anti-integrin β7 allophycocyanin (APC) (clone FIB504; Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), incubated with anti-APC-conjugated magnetic beads and separated once on the positive selection program on an Auto-MACS (Miltenyi Biotec GmbH). The positively selected lymphocytes were

91 ± 9% integrin β7-positive, whereas the remaining Chloroambucil lymphocyte population contained 36 ± 12% integrin β7+ lymphocytes as judged by fluorescence activated cell sorter (FACS) analysis. The frequency of integrin β7+ lymphocytes in the unseparated cell population was 56 ± 12%. The mucosal layers of the intestinal specimens were separated mechanically from underlying fat and muscle tissue with scissors and cut into small pieces. The mucosal fragments were incubated 4 × 15 min at 37°C on a magnetic stirrer in RPMI-1640 medium containing 10% AB-serum, 1 mM ethylenediamine tetraacetic acid (EDTA) (Sigma-Aldrich Chemie GmbH, Stienheim, Germany) and 1 mM DL-dithiothreitol (Sigma-Aldrich). Supernatants from the three first incubations, containing IEL, were poured over a nylon mesh, washed twice and kept on ice until further analysis. The remaining mucosal pieces were washed twice with Hanks’ balanced salt solution (HBSS) and were then incubated at 37°C on a magnetic stirrer in RPMI-1640 medium containing 5% AB-serum, 0·1 mg/ml DNAse 1 (D-5025; Sigma-Aldrich) and 100 U/ml collagenase (C-7657; Sigma-Aldrich) for 1·5–2 h.

Samples were extracted and run in single well qPCR reactions due to the large sample

numbers, high cost of testing, and previous work by the author’s group showing that triplicate wells give almost identical results (46). Serum samples collected at −7, −14, −21, 0, 7, 14, and 21 dpc were tested for the presence of PCV1-2 DNA and samples collected at 0, 7, 14, and 21 dpc were tested for the presence of PCV2 DNA by quantitative real-time PCR assays using primer-probe combinations as described previously (46) with the following modifications: a commercially available master mix (TaqMan Fast Universal PCR Master Mix, Applied Biosystems) was used, the reaction volume was 25 μL, only one aliquot was tested for each sample and the thermal selleck chemicals llc cycler conditions were 95°C for 2 min, followed by 40 cycles of 95°C for 10 s and 60°C for 1 min. Samples were considered negative when no signal was observed within the 40 amplification cycles. Five serial dilutions of a PCV2 genomic DNA clone (105 to 109 copies/mL) were used to generate a standard curve with a correlation coefficient of > 0.99 (46). Serum samples collected at 7, 14 and 21 dpc were tested

for the presence and amount of PRRSV RNA as described previously Crizotinib clinical trial (41). Samples were considered negative when no signal was Adenosine triphosphate observed within the 40 amplification cycles. All pigs were humanely euthanized by intravenous pentobarbital sodium overdose (Fatal-Plus, Vortech Pharmaceuticals, Dearborn, MI, USA) and necropsied at 21 dpc. The extent of macroscopic lung lesions (ranging from 0 to 100%) was estimated and scored as described previously (44). The sizes of superficial inguinal lymph nodes were compared among groups

as described previously (47). Sections of lymph nodes (superficial inguinal, external iliac, mediastinal, tracheobronchial, and mesenteric), tonsil, heart, thymus, kidney, colon, spleen, liver, small (ileum) and large intestine (spiral colon) were collected at necropsy, fixed in 10% neutral-buffered formalin, and routinely processed for histological examination. Microscopic lesions were evaluated by two veterinary pathologists (TO, PGH) who were blinded to the treatment groups. Lung sections were scored for the presence and severity of interstitial pneumonia, ranging from 0 (normal) to 6 (severe diffuse) (44). Sections of heart, liver, kidney, ileum, colon and thymus were evaluated for the presence of granulomatous inflammation and scored from 0 (none) to 3 (severe). Lymph nodes, spleen, and tonsil were evaluated based on LD and HR of follicles, ranging from 0 (normal) to 3 (severe) (22).

In contrast, IL-17A- and IL-22-secreting cells were more abundantly derived Selleckchem GS-1101 from lesional skin (Supporting Information

Fig. S3B). This observation led us to use such lesions as a source of T cells to generate CD4+ T-cell clones with various Th profiles, including Th17 and Th22 cells. Hierarchical cluster analysis performed on the cytokine pattern of skin-infiltrating T-cell clones obtained from two psoriasis patients yielded distance trees that highlighted their organization into five dominant groups, each characterized by a typical cytokine secretion profile (Fig. 3A and Supporting Information Fig. S4A). The number of clusters obtained was validated using the non-hierarchical cluster analysis (data not shown) with an excellent inter-classification comparison index (kappa agreement value κ=0.89 and 0.70 respectively). The inter-cluster differences were confirmed through the computation of the mean relative cytokine productions in each proposed cluster, followed by inter-cluster comparisons (Fig. 3B and Supporting Information Fig. S4B).

This analysis confirmed that IFN-γ was most increased in the first cluster, as compared with other clusters (p<0.0001 for both patients), IL-10 in the second cluster (p<0.0001), IL-4 (p=0.001 and p=0.0065, 1st and 2nd patient respectively) and IL-5 (p<0.0001) in the third, IL-17 selleck chemical in the fourth (p<0.0001) and IL-22 in the fifth (p<0.0001) (Fig. 3B and Supporting Information Fig. S4B). The clusters were therefore named Th1, Tr1, Th2, Th17 and Th22 respectively. Altogether, these data suggest that Th1, Th2, Tr1, Th17 and Th22 orientation can be

objectively distinguished by cluster analysis of cytokine production profiles. The Th22 subset should therefore clearly be distinguished from the previously recognized Th17 subset. We then used TCRα and TCRβ clonotypic analysis to assess whether the commitment however of these functionally distinct subsets of CD4+ T cells would be antigen-driven or TCR-independent. Surprisingly, only 45 different clonotypes were used by the 66 T-cell clones derived from the skin biopsy of a psoriasis patient. Eight different clonotypes were extensively shared between subsets and represented 39% of the T-cell infiltrate (Fig. 4). One clone was shared by four different subsets. TCR sharing between the Th17 and Th22 subset, with only one clone shared, was not more extensive than that between other subsets. TCR sharing between functionally distinct T-cell clones was confirmed in a skin biopsy from a second psoriasis patient. In this case, TCR sharing was less extensive, but clones overlapping between Th17 and Th22 as well as Th17 and Th2 were nonetheless identified among the 59 skin-derived T-cell clones analyzed (Supporting Information Fig. S4C). These results demonstrate that none of the five Th cell types use a strictly dedicated TCR repertoire.

Low quality of evidence. The true effect may be substantially different from the estimate of the effect. Very low quality of evidence. The estimate of effect is very uncertain, and often will be far from the truth. **Access to the full see more text version For a full text version of the guideline, readers need to go to the KHA-CARI website (go to the Guidelines section (http://www.cari.org.au)). “
“Advance care planning should

be available to all patients with chronic kidney disease, including end-stage kidney disease on renal replacement therapy. Advance care planning is a process of patient-centred discussion, ideally involving family/significant others, to assist the patient to understand how their illness might affect them, identify their goals and establish how medical treatment might help them to achieve these. An Advance Care Plan is only one useful outcome from the Advance Care Planning process, the education of patient and family around prognosis and treatment options is likely to be beneficial whether or not a plan is written or the individual loses decision making capacity at the end of life. Facilitating Advance Care Planning discussions requires an understanding of their purpose and communication skills

which need to be taught. Advance Care Planning needs to be supported by effective systems to enable the discussions and any resulting Plans to be used to aid subsequent decision making. “
“Albuminuria is a robust, validated cardiovascular risk

factor. It is a simple and widely available test that was Talazoparib supplier shown to be a powerful and independent predictor of prognosis in chronic heart failure. Mineralocorticoid receptor Etofibrate antagonists may reduce the acute and chronic harmful effects of mineralocorticoid receptor activation on the kidney. The objectives of the trial were to compare the effect of spironolactone versus standard acutely decompensated heart failure (ADHF) therapy on albuminuria and to investigate the role of albuminuria as a prognostic marker in patients with ADHF. Secondary analysis of a prospective, interventional study including 100 patients with ADHF. Fifty patients were non-randomly assigned to spironolactone 100 mg/day plus standard ADHF therapy (intervention group) or standard ADHF therapy alone (control group). Patients in control group were older, had higher creatinine and urea levels, and had higher proportion of microalbuminuria (all, P < 0.05). Paired comparison of baseline and day 3 log albuminuria within each group, showed a more pronounced decrease in the intervention group (1.79 ± 0.75 to 1.59 ± 0.67, P = 0.003 vs 1.89 ± 0.70 to 1.79 ± 0.74, P = 0.096). In addition, the proportion of patients with normoalbuminuria increased from baseline to day 3 in spironolactone group (20 (40%) to 27 (54%), P < 001), accordingly the number of patients in the micro and macroalbuminuria groups was reduced.

Many Māori will prefer to die at home and whānau often prefer to take their terminally ill relative home, although, as with other groups in Raf inhibitor society, the pressures of urbanization and geographical spread of modern whānau mean that this should not be assumed. When an individual prefers to die on their tūrangawaewae (tribal land) this may be geographically distant from their

current place of residence and/or rural. Good palliative care is likely to be facilitated by a heath care professional assisting the patient and whānau with finding appropriate health care services in their chosen place of death, for example identifying a local general practitioner and referring to local palliative care services. Community palliative care services may be more acceptable than inpatient hospice care to many Māori. In hospital or hospice, whānau and patients should

be offered a single room and access to appropriate spiritual and cultural support. As autopsy can be particularly distressing to Māori it is appropriate to prepare whānau in advance if referral to the coroner and/or autopsy is likely to be necessary and explain learn more why.[9] Care of the tūpāpaku (deceased) can be a particularly sensitive area as it is generally highly ritualized in Māori culture. Whānau may have specific cultural and spiritual practices they wish to observe around handling of the body, including washing and dressing and staying with the tūpāpaku as they progress from the ward, to the mortuary and to the funeral director then marae. The way in which the tūpāpaku is transported is also significant to many Māori, for example wrapped in allocated linen, feet first and following a pre-determined route away from public thoroughfares. Blessing the room the tūpāpaku died in with a karakia prior to cleaning may also be appropriate. Non-specific serine/threonine protein kinase Again seeking advice from local kaumātua and specifically asking whānau is likely to be the best way to

avoid causing inadvertent offense by breaching protocol.[9] Individual patients and whānau may wish to use rongoā (traditional Maori methods of healing) to achieve their goals of care. Considering the Whare Tapa Whā model, rongoā may be valued for their contribution to aspects of well-being other than physical health. Local kaumātua (elders) can advise on local practice. The handling of food, taonga (valuables), the head and human waste are areas to be aware of. Generally, food and medicines for human consumption should be kept separate from items for general use, for example microwaves or refrigerators should be used for either food preparation/storage or non-food uses (e.g. heating wheat bags), not both, tea towels should only be used for drying dishes and tables should not be sat on.

The study was approved by local Ethics Committees and informed consent was obtained from the donors. In the following sections references

are given to papers which have used the same samples for other purposes. A recombinant fragment of MASP-1 comprising the CCP1-CCP2-SP domains (rCCP1-CCP2-SP) was produced in Escherichia coli, and refolded and purified as described previously [13]. A synthetic peptide representing the 15 C-terminal amino acid residues of human MASP-1 (CHHNKDWIQRVTGVR) was coupled to keyhole limpet haemocyanin. Three Wistar rats were immunized four times subcutaneously with 10 µg of this conjugate, emulsified first in complete Freund’s adjuvant and then in incomplete Freund’s adjuvant for boosts. Sera from the animals were tested for reactivity towards SAHA HDAC research buy rCCP1-CCP2-SP coated onto microtitre wells. All rats responded and the rat with the highest titre was selected. IgG was purified from the anti-serum by affinity chromatography on Protein G-coupled beads. The serum was diluted 1/1 in phosphate-buffered saline (PBS; 137 mM NaCl, 2·7 mM KCl, 1·5 mM KH2PO4, 8·1 mM Na2HPO4, pH 7·4) selleck chemicals llc containing 10 mM EDTA (PBS/EDTA), and passed through the beads. After washing, the

bound IgG was eluted with 0·1 M glycine, pH 2·4. The immunoglobulin concentration was determined by spectroscopy at 280 nm. The purified IgG was biotinylated by standard procedure [26] using 167 µg biotin-N-hydroxysuccinimide ester (Sigma-Aldrich, Bumetanide St Louis, MO, USA) per mg antibody. The anti-MASP-1 anti-serum was tested by Western blotting. MBL/MASP complexes were purified from serum by affinity chromatography on mannan coupled to Sepharose beads, as described previously [8]. The complexes, as well as a preparation of rCCP1-CCP2-SP, were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in non-reducing conditions followed by blotting onto a membrane. For the preparation of Western blotting strips, MBL/MASP

complexes corresponding to 30 µg MBL were loaded onto a single-well XT-Criterion pre-cast 4–12% gradient Bis-Tris polyacrylamide gel (Bio-Rad, Copenhagen, Denmark) and cut into 2·5-mm-wide strips after blotting, resulting in approximately 1 µg MBL (+ associated proteins) per strip. The proteins were blotted onto a nitrocellulose membrane (Hybond-ECN; GE Healthcare, Hilleroed, Denmark) in transfer buffer (25 mM Tris, 0·192 M glycine, 20% v/v ethanol, 0·1% w/v SDS, pH 8·3) for 500 volt-hours. The membrane was blocked in 0·1% Tween 20 in Tris-buffered saline (TBS) (10 mM Tris–HCl, 140 mM NaCl, 1·5 mM NaN3, pH 7·4) before being cut into strips. The strips were incubated with primary antibodies (normal rat IgG or rat anti-MASP-1 antibody) diluted in primary buffer (TBS with 0·05% Tween 20 (TBS/Tw), 1 mM EDTA, with 1 mg human serum albumin (HAS) and 100 µg normal human IgG (hIgG) added per ml) in eight-well trays (Octaline, Pateof, Denmark) for 2·5 h on a rocking table.