Citrate buffer

After dewaxing, brain sections were boiled (in an 850 W microwave oven) for 15 min in citrate buffer (2.1 g citric acid monohydrate/l, pH = 6) (Carl Roth, Karlsruhe, Germany). Endogenous peroxidase was inhibited by 1% H₂O₂ in pure methanol (Merck, Darmstadt, Germany) for 15 min. Sections were incubated with 10% normal pig serum (Biochrom, Berlin, Germany) to block nonspecific binding of immunoglobulins and then with the polyclonal antibody CTGF (1:100, BMA; USbio, Swampscott, Massachusetts, USA) overnight at 4°C. Antibody binding to tissue sections was visualized with a biotinylated rabbit anti-mouse IgG F(ab)₂ antibody fragment (1 : 400; Dako, Hamburg, Germany). Subsequently, sections were incubated with a horseradish peroxidase-conjugated streptavidin complex for 30 min (1: 100; Dako), followed by development with diaminobenzidine substrate (Fluka, Neu-Ulm, Germany). Finally, sections were counterstained with Mayer’s hemalaun. As negative controls, the primary antibodies were excluded.

For immunohistochemistry, we cut paraffin-embedded tissue specimens into 3 µm thick slices, dewaxed them twice in xylol (Carl Roth, Karlsruhe, Germany), and rehydrated them in a graded series of ethanol (Carl Roth, Karlsruhe, Germany) diluted in distilled water (100%, 96%, 70%, only distilled water). Afterwards, we boiled the specimens for 10 min at 120 °C in 20 mM citrate buffer (pH = 6.0) (Carl Roth, Karlsruhe, Germany) before treating the slices with 0.7% hydrogen peroxide (Carl Roth, Karlsruhe, Germany) and 10% normal goat serum (Invitrogen, Waltham, MA, USA). Then, we stained BRMS1 using the Envision System HRP DAB (DAKO, Jena, Germany) and the anti-BRMS1 antibody ab65244 (Abcam, Cambridge, UK) in a 1:1500 dilution, according to the manufacturer’s instructions. Finally, we counterstained the cells’ nuclei with hemalaun solution acid according to Mayer (Roth, Karlsruhe, Germany) and embedded the specimens in a xylol-based mounting medium (ORSAtec, Bobingen, Germany).