Anti ndufs4

OXPHOS complexes (complex I-V) and proliferation indices (Ki-67 and PHH3) were evaluated in deparaffinized tumor sections of NB xenografts. For immunohistochemical staining the following antibodies were used: anti-NDUFS4 (1:1000, Abcam), anti-SDHA (1:2000, MitoSciences), anti-UQCRC2 (1:1500, MitoSciences), anti-MTCO2 (1:1000, MitoSciences), anti-ATP5A (1:2000, MitoSciences), and anti-VDAC1 (Voltage-dependent anion-selective channel protein 1; 1:3000, MitoSciences), anti-Ki-67 (1:200, Dako) and anti-PHH3 (1:500, Cellmarque). All antibodies were diluted in Dako antibody diluent with background-reducing components (Dako). The staining and scoring procedures for the OXPHOS complexes were performed as reported previously [17 (link), 45 (link)]. Proliferation was scored by evaluating the proportion of positively stained nuclei, scoring at least 500 cells per slide (Ki-67) or counting the number of positively stained nuclei per high power field (PHH3). For all tumors at least five representative regions were scored, magnification was 400-fold.

600g homogenates were used for western blot analysis as described before [3 (link)]. In brief, a total of 10 μg protein was separated on 10% acrylamide/bis-acrylamide gels and transferred to nitrocellulose membranes (Amersham Biosciences) using a CAPS buffer (10 mM 3-[cyclohexylamino]-1-propane sulfonic acid, pH 11; 10% methanol). The following primary antibodies were diluted in 1% western blocking reagent (Roche Diagnostics) dissolved in TBS-T: anti-GAPDH (Glyceraldehyde-3-phosphate dehydrogenase, 1:10000, Trevigen), anti-NDUFS4 (NADH dehydrogenase [ubiquinone] iron-sulfur protein 4; 1:2000, Abcam), anti-SDHA (Succinate dehydrogenase [ubiquinone] flavoprotein subunit; 1:5000, Abcam), anti-UQCRC2 (Cytochrome b-c1 complex subunit 2; 1:1000, Sigma), anti-MTCO2 (Cytochrome c oxidase subunit 2; 1:5000, Abcam), anti-ATP5A (ATP synthase subunit alpha; 1:1000, Protein Tech), anti-SCOT (Succinyl-CoA:3-ketoacid coenzyme A transferase 1; 1:1000, Abnova). Horseradish peroxidase-labeled secondary antibodies were used at a dilution of 1:1000 (Dako) and detection was carried out with Lumi-Light POD-substrate (Roche).