Immunohistochemical staining for type I collagen (Col-I) was performed using an affinity-purified goat anti-mouse Col-I antibody (Southern Biotechnology Associates) as described previously.
MALDI-IMS/MSI were performed using a Q-Star Pulsar i hybrid quadrupole time-of-flight mass spectrometer (Applied Biosystems/MDS Sciex, Concorde, Ontario, Canada) fitted with a variable repetition rate Nd YV04 laser set at 5 kHz.
b a DWI RMI scan, performed using the ADC (Apparent Diffusion Coefficient) protocol, which shows no alteration of signal.
Relative quantification of the steady-state transcript levels of the nDNA- and mtDNA-encoded subunits of complex I was performed using Real time PCR.
The long-term confocal imaging of aggregates in cells treated with cucurbitacin I was performed using a Pathology Devices stage incubator (maintaining 37°C and 5% CO2) on a Nikon A1R confocal microscope.
Immunohistochemistry for collagen I was performed using the SABC kit (Maxim, Fuzhou, China) according to the manufacturer's instructions.
Proteins were visualized using colloidal Coomassie R250, or the gels were destained with MilliQ water for in-gel complex I assays performed using 120 µ m NADH and 0.5 mg ml−1 nitro blue tetrazolium (NBT) (55).
The validation of the IDS-iSYS IGF-I immunoassay (IDS, Boldon, UK) for measurement of rabbit IGF-I was performed using commercially available rabbit serum samples (Charles-River, Sulzfeld, Germany).
After cell disruption and a pre-clearing step, pull-down of biotin-tagged RIG-I was performed using streptavidin sepharose beads (GE Healthcare).
