PMC:13902 / 19239-21652 JSONTXT

{"project":"Colil","denotations":[{"id":"T52","span":{"begin":1256,"end":1258},"obj":"2465297"},{"id":"T53","span":{"begin":1259,"end":1261},"obj":"1764993"},{"id":"T54","span":{"begin":1262,"end":1264},"obj":"2450577"},{"id":"T55","span":{"begin":1022,"end":1024},"obj":"2350783"},{"id":"T56","span":{"begin":617,"end":619},"obj":"2465297"},{"id":"T57","span":{"begin":941,"end":943},"obj":"1764993"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/docs/sourcedb/PubMed/sourceid/"}],"text":"Immunohistochemistry of TGF-βs\nImmunohistochemical staining was done using rabbit polyclonal antibodies raised against synthetic peptides that correspond to regions in the mature forms of TGF-β1, TGF-β2, and TGF-β3. Antibodies to TGF-β1 were raised against a synthetic peptide corresponding to residues 1-30 of the mature protein in either the Laboratory of Chemoprevention (anti-TGF-β1-LC; NIH, Bethesda, MD, USA) or the Collagen Corporation (anti-TGF-β1-CC; Palo Alto, CA, USA). These antibodies were raised against different preparations of the 1-30 peptide, and they recognize different epitopes of this peptide [19]. The LC antibody usually stains intracellular TGF-β1, whereas the CC antibody stains extracellular TGF-β1. Anti-TGF-β2 (sc-90; Santa Cruz Biotechnologies Inc) was raised to a peptide corresponding to residues 72-99 of the mature TGF-β2. Anti-TGF-β3 (anti-50-60-β3-LC) was raised against residues 50-60 of mature TGF-β3 [20]. Anti-LTBP (Ab39) was raised against the purified full-length platelet LTBP [21].\nThe antibodies were affinity purified against the immunizing peptide (anti-TGF-β3) or against protein A sepharose (anti-TGF-β1-LC, anti-TGF-β1-CC, and anti-TGF-β2), and have been assayed for specificity by Western blot analysis [19,20,33]. All antibodies reacted with the appropriate TGF-β isoform except anti-TGF-β1-CC, which showed some cross-reactivity with TGF-β3 on Western blots.\nImmunohistochemical staining was performed using an indirect immunoperoxidase detection protocol (Vectastain Elite kit; Vector Laboratories) following treatment of sections with hyaluronidase to improve antibody penetration. Optimal antibody concentrations were determined by titration on select samples before analysis of the full experimental set. Staining was shown to be specific in control experiments in which either the primary antibody was preincubated with a 50-fold molar excess of immunizing peptide before being applied to the section (anti-TGF-β2, anti-TGF-β3), or the section was stained with an equivalent concentration of nonimmune rabbit immunoglobulin (anti-TGF-β1-LC, anti-TGF-β1-CC, and anti-LTBP). In analysis of the full experimental set, for any given antibody all sections were stained at the same time so as to be directly comparable, and a normal mouse embryo section was included as a positive control. A normal rabbit immunoglobulin control was also run for the whole set."}