PMC:17827 / 18874-22631 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/17827","sourcedb":"PMC","sourceid":"17827","source_url":"http://www.ncbi.nlm.nih.gov/pmc/17827","text":"Immunohistochemistry\nImmunohistochemical analysis was performed on cryostat sections of RASM (fixed with acetone for 10 min at 4°C) or chamber slides (fixed with 100% methanol for 10 min at room temperature; Nunc, Wiesbaden, Germany) using the antibodies presented in Table 3. The mouse mAbs, diluted in Tris–buffered saline (TBS)/1% bovine serum albumin for per-oxidase/alkaline phosphatase detection, were added for 30 min in a humid chamber at room temperature following pre-incubation of the sections/chambers with 20% normal human serum in TBS for 20 min (pH 7.4). A peroxidase-coupled rabbit anti-mouse antibody (Dako) and a peroxi-dase-coupled swine anti-rabbit antibody (Dako; both 1:30 in TBS/20% human serum) were added for 30 min each. The peroxidase was revealed using diaminobenzidine (5 min, 0.5 mg diaminobenzidine in 1 ml PBS [pH 7.4] containing 30 μl H2O2). Blocking of endogenous peroxidase (0.03% H2O2) before immunohistochemical analysis, as performed in some experiments, did not alter the staining results.\nAlternatively, either the indirect alkaline phosphatase or the alkaline phosphatase anti-alkaline phosphatase (APAAP) techniques were used for detection of the monoclonal or polyclonal primary antibodies [35]. An alkaline phosphatase-coupled goat-anti-mouse antibody (Dako) was applied for 30 min (1:30 in TBS/20% human serum; pH 7.4) in the case of the indirect alkaline phosphatase, and the alkaline phosphatase was revealed in substrate buffer (TBS; pH 9.5) containing 0.16 mg/ml 5-chromo-4-chloro-3-indoxylphosphate and 0.15 mg/ml nitroblue tetra-zolium chloride (both from Boehringer Mannheim). In the case of the APAAP, a rabbit anti-mouse antibody (Dako) was applied for 45 min (1:100 in TBS/20% human serum; pH 7.4). The sections/chamber slides were washed, incubated for 45 min with APAAP complex (1:80 in TBS/1% bovine serum albumin; Dianova), and washed again with TBS. To amplify the staining, incubation with the rabbit anti-mouse mAb and the APAAP was repeated once. The alkaline phosphatase was revealed in Tris–HCl buffer (pH 8.6) containing 0.3 mg/ml Naphthol AS-MX phosphate (first dissolved at 15 mg/ml in dimethylformamide; both from Sigma), 1 mg/ml Fast Red TR salt or Fast Blue BB salt, and 0.24 mg/ml levamisole (all from Sigma). The solution was filtered and added to the sections/chamber slides for 30 min in a humid chamber. For the APAAP technique, polyclonal primary antibodies (against procollagen I and III) were incubated with a secondary mouse anti-rabbit antibody (Dako) before using the secondary detection systems.\nDouble-staining experiments on cryostat sections of the RA SM were performed by combining the peroxidase technique with the APAAP technique. The sections were blocked with 20% normal human serum in TBS for 30 min following completion of peroxidase staining. Isotype-matched mouse mAbs, rabbit sera, or purified rabbit immunoglobulins at identical concentrations to the primary antibodies were used as controls for all immunohistochemical analyses (Table 3), which gave no positive results. Cross-reactivity of the peroxidase and the alkaline phosphatase detection (theoretically possible on the basis of employing mouse primary mAbs and anti-mouse secondary antibodies in both systems) was never observed when using control Ig or when omitting one or both primary mAbs, presumably because the peroxidase reaction with diaminobenzidine eliminates all immunoreactive epitopes from this step.\nFor comparative immunohistochemical and FACS analyses, the percentage of cells stained for the individual markers following immunohistochemistry on chamber slides was determined (total of 100 cells analyzed) and compared with the percentage of positive cells in FACS analysis (see later).","divisions":[{"label":"Title","span":{"begin":0,"end":20}}],"tracks":[{"project":"2_test","denotations":[{"id":"11178129-2212003-4386854","span":{"begin":1234,"end":1236},"obj":"2212003"}],"attributes":[{"subj":"11178129-2212003-4386854","pred":"source","obj":"2_test"}]},{"project":"Colil","denotations":[{"id":"T61","span":{"begin":1234,"end":1236},"obj":"2212003"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/docs/sourcedb/PubMed/sourceid/"}],"attributes":[{"subj":"T61","pred":"source","obj":"Colil"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#b7ec93","default":true},{"id":"Colil","color":"#ec93d1"}]}]}}