Immobilon hrp substrate reagents

Tissue for western blotting was obtained from the New York Brain Bank frozen, and then rapidly homogenated at 4 °C in lysis buffer containing 50 mM of Tris-HCl, 150 mM of NaCl, 5 mM of EDTA and 1% of Triton-Tx. Protein concentration of the total homogenate was measured using the BCA assay (Thermo Scientific). 60 μg of protein per lane were run on a 10% polyacrylamide gel for HLA antibody and on a 15% polyacrylamide gel for β2m antibody. Proteins were transferred to PVDF membranes (Immobilon, Millipore Corporation, Bedford, MA, USA). After blocking in 5 % dry milk, primary antibodies were applied overnight at 4 °C, then rinsed 3 times in Tris-buffered saline containing 0.1% Tween-20 (Fisher Scientific). Peroxidase-labeled secondary antibodies were applied for 1 h at RT, blots rinsed in Tris-buffered saline-Tween as before, developed with enhanced chemiluminescence (Immobilon HRP substrate reagents, Millipore), and then exposed to film (Crystalgen). HLA (1:100, from Santa Cruz Biotechnologies) and β2m (1:100, from Abnova) primary antibodies were used. Actin (1:500,000, from Sigma Aldrich) primary antibody was used as a loading control.

The brains of 3 C57/BL6 and 3 B6.129P2-B2m^tm1Unc/J were dissected on ice and then rapidly homogenated at 4 °C in lysis buffer containing 50 mM of Tris-HCl, 150 mM of NaCl, 5 mM of EDTA and 1% of Triton-Tx. Protein concentration of the total homogenate was measured using the BCA assay (Thermo Scientific). 20 μg of protein per lane were run on a 12% polyacrylamide gel. Proteins were transferred to PVDF membranes (Immobilon, Millipore Corporation, Bedford, MA, USA). After blocking in 5 % dry milk, primary antibody (MHC-I antibody raised against the H-2K^b and H-2D^b class I alloantigens, mouse monoclonal, 1:100, BD Biosciences), was applied overnight at 4 °C, then rinsed 3 times in Tris-buffered saline containing 0.1% Tween-20 (Fisher Scientific). Peroxidase-labeled secondary antibodies were applied for 1 h at RT, blots rinsed in Tris-buffered saline-tween as before, developed with enhanced chemiluminescence (Immobilon HRP substrate reagents, Millipore), and then exposed to film (Crystalgen). Actin (1:500,000, from Sigma Aldrich) primary antibody was used as a loading control. 20 μg of mouse spleen homogenate was loaded as a positive control for the MHC-I antibody.