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Toluidine Blue Staining (toluidine + blue_staining)
Selected AbstractsSmoking delays chondrogenesis in a mouse model of closed tibial fracture healingJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 12 2006Hossam B. El-Zawawy Abstract Smoking delays the healing process and increases morbidity associated with many common musculoskeletal disorders, including long bone fracture. In the current study, a murine model of tibial fracture healing was used to test the hypothesis that smoking delays chondrogenesis after fracture. Mice were divided into two groups, a nonsmoking control group and a group exposed to cigarette smoke for 1 month prior to surgical tibial fracture. Mice were euthanized at 7, 14, and 28 days after surgery. The outcomes measured were immunohistochemical staining for type II collagen protein expression as a marker of cartilage matrix and proliferating cell nuclear antigen (PCNA) staining to measure proliferation at the site of injury. Toluidine blue staining and histomorphometry were used to quantify areas of cartilaginous and noncartilaginous fracture callus. Radiographs were analyzed for evidence of remodeling after injury. At day 7 after injury, mice exposed to cigarette smoke had a smaller fracture callus with less cartilage matrix compared to controls. Proliferation was present at high levels in both groups at this time point, but proliferating cells had a more immature morphology in the smoking group. At day 14, chondrogenesis was more active in smokers compared to controls, while a higher percentage of bone was present in the control animals. At day 28, X-ray analysis revealed a larger fracture callus remaining in the smoking animals. Together, these findings show that the chondrogenic phase of tibial fracture healing is delayed by smoking. This study represents, to our knowledge, the first analysis of molecular and cellular mechanisms of healing in a smoking mouse fracture model. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res [source] Comparison of Four Staining Methods for Detection of Mast Cells in Equine Bronchoalveolar Lavage FluidJOURNAL OF VETERINARY INTERNAL MEDICINE, Issue 2 2006Mathilde Leclere Mast cells normally are present in equine bronchoalveolar lavage fluid (BALF), but usually represent <2% of all cells in healthy horses. An increased percentage of mast cells has been associated with airway hyperactivity and inflammatory airway diseases, but marked differences are reported between studies in normal and diseased horses. Because an abnormal mast cell count may be of clinical relevance, we compared the ability of a fast Romanowsky method to stain mast cell granules with that of 3 metachromatic stains: automated Romanowsky, May-Grünwald Giemsa, and toluidine blue stains. The BALF cells from 24 horses were studied. A differential cell count was performed blindly on 400 cells. The percentages of mast cells obtained were analyzed by means of repeated-measures analysis of variance and Fischer's PLSD test. The Bland and Altman method was used to assess agreement among stains. The mean percentage of mast cells in BALF was significantly lower with the fast Romanowsky than with the automated Romanowsky, May-Grünwald Giemsa, and toluidine blue stains. With the fast Romanowsky stain, the metachromatic granules of mast cells were not stained, and their identification was based on morphologic criteria. Toluidine blue staining allowed detection of the highest mean percentage of mast cells, but was inadequate for performing a differential cell count on other cell types. In conclusion, fast Romanosky stain may be inadequate for detection of mast cells in equine BALF, whereas automated Romanowsky, May-Grünwald Giemsa, and toluidine blue stains provide metachromatic staining of mast cell granules. [source] Sensitive fluorescence detection of polyphosphate in polyacrylamide gels using 4,,6-diamidino-2-phenylindolELECTROPHORESIS, Issue 19 2007Stephanie A. Smith Dr. Abstract PAGE is commonly used to identify and resolve inorganic polyphosphates (polyP). We now report highly sensitive and specific staining methods for polyP in polyacrylamide gels based on the fluorescent dye, 4,,6-diamidino-2-phenylindol (DAPI). DAPI bound to polyP in gels fluoresced yellow while DAPI bound to nucleic acids or glycosaminoglycans fluoresced blue. Inclusion of EDTA prevented staining of glycosaminoglycans by DAPI. We also identified conditions under which DAPI that was bound to polyP (but not nucleic acids or other anionic polymers) rapidly photobleached. This allowed us to develop an even more sensitive and specific negative staining method that distinguishes polyP from nucleic acids and glycosaminoglycans. The lower LOD using DAPI negative staining was 4,pmol (0.3,ng) phosphate per band, compared to conventional toluidine blue staining with a lower LOD of 250,pmol per band. [source] Expression of COX-1, COX-2 and inducible nitric oxide synthase in superficial gastritis, mucosal dysplasia and gastric carcinomaJOURNAL OF DIGESTIVE DISEASES, Issue 3 2001Yuqin Luo OBJECTIVE: To investigate the significance of the expression of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in superficial gastritis, gastric mucosal dysplasia and gastric carcinoma, and to study the relationship between COX-2, iNOS, gastric carcinoma and Helicobacter pylori infection. METHODS: Polyclonal antibodies to COX-1, COX-2 and iNOS were used detect their expression and the status of H. pylori infection in 92 specimens of paraffin-embedded gastric tissue. Of the 92 patients, 33 had superficial gastritis, 30 had gastric mucosal dysplasia and 29 had gastric cancer. Helicobacter pylori was detected by toluidine blue staining. RESULTS: Expression of COX-2 and iNOS in gastric cancer (65.5%, 62.1%) was significantly higher than that in gastritis (18.2%, 18.2%; P < 0.01). Expression of COX-2 and iNOS in gastritis with H. pylori infection was higher than that in gastric mucosal dysplasia with H. pylori infection. The expression of COX-2 and iNOS occurred concomitantly in gastritis, dysplasia and gastric cancer. CONCLUSION: Inflammation and H. pylori infection may be able to stimulate the expression of COX-2 and iNOS, which might be involved in gastric carcinogenesis. [source] A simple protocol for paraffin-embedded myelin sheath staining with osmium tetroxide for light microscope observationMICROSCOPY RESEARCH AND TECHNIQUE, Issue 7 2008Federica Di Scipio Abstract Experimental investigation of peripheral nerve fiber regeneration is attracting more and more attention among both basic and clinical researchers. Assessment of myelinated nerve fiber morphology is a pillar of peripheral nerve regeneration research. The gold standard for light microscopic imaging of myelinated nerve fibers is toluidine blue staining of resin-embedded semithin sections. However, many researchers are unaware that the dark staining of myelin sheaths typically produced by this procedure is due to osmium tetroxide postfixation and not due to toluidine blue. In this article, we describe a simple pre-embedding protocol for staining myelin sheaths in paraffin-embedded nerve specimens using osmium tetroxide. The method involves immersing the specimen in 2% osmium tetroxide for 2 h after paraformaldeyde fixation, followed by routine dehydration and paraffin embedding. Sections can then be observed directly under the microscope or counterstained using routine histological methods. Particularly good results were obtained with Masson's trichrome counterstain, which permits the imaging of connective structures in nerves that are not detectable in toluidine blue-stained resin sections. Finally, we describe a simple protocol for osmium etching of sections, which makes further immunohistochemical analysis possible on the same specimens. Taken together, our results suggest that the protocol described in this article is a valid alternative to the conventional resin embedding-based protocol: it is much cheaper, can be adopted by any histological laboratory, and allows immunohistochemical analysis to be conducted. Microsc. Res. Tech., 2008. © 2008 Wiley-Liss, Inc. [source] Expression of Oestrogen Receptor , During Development of the Skeleton in Mice Fetuses: Immunohistochemical StudyANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 6 2008E. Lovsin Barle Summary Sequential pattern of ossification and expression of oestrogen receptor , (ER,) during development of the skeleton in male and female mice fetuses was investigated. Twenty-seven mice fetuses of gestational age between 14.5 and 18.5 days post coitum (p.c.) were examined by haematoxylin,eosin and toluidine blue staining to determine the ossification. The presence of ER, was detected by immunostaining using ER,-specific antibodies. Ossification centres were determined in fetuses of 14.5 days p.c. of both sexes in the base of skull, ribs and front limbs, while in the mandible ossification was observed only in female fetuses at that age. ER, was found in all investigated tissues in which the occurrence of ossification centres was determined. ER, was first detected in some tissues involved in ossification at 14.5 days p.c. in fetuses of both genders. There were some minor gender differences in the pattern of ER, expression. ER, was localized in the metatarsal chondrogenic condensations at 14.5 days p.c. and in phalangeal chondrocytes at 17.5 and 18.5 days p.c. only in females. ER,-positive osteogenic cells at 14.5 days p.c. in the mandible were seen only in females. At 16.5 days p.c. male but not female fetuses expressed ER, in the vertebrae. Our findings support the view that ER, protein is found in the tissues that undergo bone formation and that ER, expression in these tissues shows only minor gender differences in mice fetuses. [source] Lesional and nonlesional skin from patients with untreated juvenile dermatomyositis displays increased numbers of mast cells and mature plasmacytoid dendritic cellsARTHRITIS & RHEUMATISM, Issue 9 2010Sheela Shrestha Objective To investigate the distribution of mast cells and dendritic cell (DC) subsets in paired muscle and skin (lesional/nonlesional) from untreated children with juvenile dermatomyositis (DM). Methods Muscle and skin biopsy samples (4 skin biopsy samples with active rash) from 7 patients with probable/definite juvenile DM were compared with muscle and skin samples from 10 healthy pediatric controls. Mast cell distribution and number were assessed by toluidine blue staining and analyzed by Student's t -test. Immunohistochemical analysis was performed to identify mature DCs, myeloid DCs (MDCs), and plasmacytoid DCs (PDCs) by using antibodies against DC-LAMP, blood dendritic cell antigen 1 (BDCA-1), and BDCA-2, respectively. Myxovirus resistance protein A (MxA) staining indicated active type I interferon (IFN) signaling; positive staining was scored semiquantitatively and analyzed using the Mann-Whitney U test. Results Both inflamed and nonlesional skin from patients with juvenile DM contained more mast cells than did skin from pediatric controls (P = 0.029), and comparable numbers of mast cells were present in lesional and nonlesional skin. Interestingly, mast cell numbers were greater in skin than in paired muscle tissue from patients with juvenile DM (P = 0.014) and were not increased in muscle from patients with juvenile DM compared with control muscle. Both muscle and skin from patients with juvenile DM showed more mature PDCs and MxA staining than did their corresponding control tissues (P < 0.05). In both muscle and skin from patients with juvenile DM and in pediatric control muscle, there were fewer MDCs than PDCs, and the distributions of MDCs and PDCs were similar in pediatric control skin samples. Conclusion The identification of mast cells in skin (irrespective of rash) from patients with juvenile DM, but not in paired muscle tissue, suggests that they have a specific role in juvenile DM skin pathophysiology. In skin from patients with juvenile DM, increased numbers of PDCs and increased expression of type I IFN,induced protein suggest a selective influence on T cell differentiation and subsequent effector function. [source] | |||||||||||||