Fig. 6.

Synovial tissue (ST) angiogenesis, oxidative stress and cellular bioenergetics. To support the concept that oxidative stress, angiogenesis and energy metabolism are interconnected processes that co-exist during the inflammation milieu, double-immunofluorescence staining was performed. ST slides were co-incubated with primary mouse antibody against human 4-hydroxy-2-nonenal (4-HNE) and with primary rabbit antibodies against angiogenic factors (vascular endothelial growth factor [VEGF], angiopoietin 2 [Ang2], tyrosine kinase receptor [Tie2]), glycolytic proteins (glyceraldehyde 3-phosphate dehydrogenase [GAPDH], pyruvate kinase isozyme 2 [PKM2], glucose transporter 1 [GLUT1]) and a mitochondrial marker (adenosine triphosphate synthase subunit β [ATP5B]). Representative merged immunofluorescence images demonstrate examples of co-localisation (yellow) of 4-HNE with VEGF, Ang2, Tie2, GAPDH, PKM2, GLUT1 and ATP5B. Cells stained green are positive for 4-HNE only; cells stained red are positive only for VEGF, Ang2, Tie2, GAPDH, PKM2, GLUT1 and ATP5B. Arrows indicate examples of co-localisation. Magnification of photomicrographs × 20, insets show high-power magnification of co-localisation. Representative images show single immunofluorescence of 4-HNE, VEGF, Ang2, Tie2, GAPDH, PKM2, GLUT1 and ATP5B along with their controls. Isotype-matched antibodies are shown in Additional file 3: Figure S3 and Additional file 4: Figure S4