Western blot analysis of TNFα and its receptors

Mouse corneas were homogenized in lysis buffer containing 100mM Tris-HCl and SDS 1%. Lysates were separated by 12% SDS-PAGE and transferred to a PVDF membrane. After blocking in 5% non-fat dried milk powder in TBS/Tween 0.05% 1 h at room temperature, the membranes were incubated overnight at 4°C with primary antibody anti-TNFα (1:500; Cat # ABIN677318; antibodies-online Inc., USA), anti-TNF-Rp55 (1:100; Cat # AP06465PU-N; Acris) and anti-β-Actin (1:10000, Cat # ab8224, abcam), diluted in TBS/Tween 0.05%. Membranes were washed three times for 10 minutes in TBS/Tween 0.05% and then incubated with the appropriate anti-rabbit or anti-mouse IgG HRP-labelled antibody (1:5000) (GE Healthcare UK Limited, Buckinghamshire, UK) for 1 h at room temperature. Proteins were visualized via chemiluminescence reaction (Bio-Rad Laboratories, Hercules, CA, USA). Blots were digitalized using a ChemiDoc YRS Imager with the software QuantityOne (Bio-Rad, Laboratories,Germany) and densitometry was performed with ImageJ (NIH, USA).