Anti lemd1 antibody

Whole-cell lysate was obtained using M-PER mammalian protein extraction reagent (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer's protocol, and 50 μg of the lysate was subjected to immunoblotting in 12.5% SDS–PAGE, followed by electrotransfer to polyvinylidene fluoride membranes (Thermo Fisher Scientific). The membranes were incubated with anti-LEMD1 antibody (Abcam) and then with peroxidase-conjugated IgG (Medical & Biological Laboratories). The immune complex was visualised by ECL western blotting detection system (GE Healthcare, Amersham place, UK). Anti-GAPDH antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, clone V-18) was used as an internal control.

Consecutive 3-μm sections were cut from each block and immunohistochemistry was performed and antigen retrieval by microwaving in citrate buffer at 95 °C for 30 min. After endogenous peroxidase blocking with 3% H₂O₂-methanol, specimens were incubated in a 10% skim milk solution (Morinaga Milk, Tokyo, Japan) for 20 min to block non-specific antibody reactions. Anti-LEMD1 antibody (Abcam, Cambridge, UK; polyclonal, dilution at 1 : 50) was applied as the primary antibody for 2 h, followed by incubation with the secondary antibody peroxidase-conjugated anti-mouse or rabbit (Medical & Biological Laboratories, Nagoya, Japan; dilution at 1 : 200) for 30 min at room temperature. The specimens were colour-developed with diaminobenzidine solution (Dako, Carpinteria, CA, USA) and specimens were counterstained with Meyer's hematoxylin (Sigma-Aldrich Corporation, St Louis, MO, USA). Immunoreactivity was classified according to Allred's score (AS) (Allred et al, 1998 (link)). We divided immunoreactivity into three criteria based on AS according to our prior report (Kurihara et al, 2013 (link)): negative, AS of 0; LEMD1 low, AS of 2∼4; LEMD1 high, AS of 5∼8.