Rabbit anti hmgb1 polyclonal antibody

Immunohistochemical (IHC) staining procedure was performed as previously described [22 (link)]. Tissue sections were incubated overnight in rabbit anti-HMGB1 polyclonal antibody (1:300, Abcam, Cambridge, U.K.) at 4°C and incubated with an anti-rabbit secondary antibody (1:1000, Abcam, Cambridge, U.K.) for 30 min at 37°C on the following day. Based on the staining intensity and the proportion of positive cells, IHC scores for HMGB1 were measured by two pathologists with no prior knowledge of clinicopathological results, and any discrepancies in scores were discussed until a consensus was reached. Five visual field (10*40) were chosen randomly and every case was scored by combining intensity points and proportion points. Proportion of positive cells were classified into four categories: 1 point (≤25%), 2 points (26–50%), 3 points (51–75%), and 4 points (>75%). Then the intensity of nuclear or cytoplasmic staining were scored as follows: weak staining detectable above background scored 1, moderate staining scored 2, and strong staining scored 3. We took the mean scores, which combined intensity points with proportion points of five visual fields as the final scores. Cases with final scores over 4 were regarded as high expressions of HMGB1

Western blot analyses were performed on retinal extracts of the retina, which were homogenized in ice-cold lysis buffer (1% sodium dodecyl sulfate, 1.0 mM sodium orthovanadate, 10 mM Tris, pH 7.4). Aliquots of lysed tissue, each containing 50 μg of total protein were heated at 100°C for 10 min with an equivalent volume of 2× sample buffer and were loaded onto 10% polyacrylamide gels. Proteins were electrophoresed and subsequently blotted onto a polyvinylidene fluoride membrane. The membrane was blocked with 5% non-fat dry milk dissolved in 0.01 M PBS (pH 7.4) containing 0.05% Tween-20 for 1 h at room temperature. The membrane was then incubated for 15 h at 4°C with rabbit anti-HMGB1 polyclonal antibody (1:1000; Abcam) in blocking solution. The membrane was rinsed 3 times with PBS containing 0.05% Tween-20 (10 min per wash), and was then incubated with peroxidase-conjugated donkey anti-goat IgG antibody (1:1000; Jackson ImmunoResearch) for 2 h at room temperature. Blots were developed using the Enhanced Chemiluminescence Detection Kit (Amersham, Arlington Heights, IL, United States) and densitometry was performed using the Eagle Eye TMII Still Video System (Stratagene, La Jolla, CA, United States).

Purified HMGB1 and desHMGB1 were subjected to SDS-PAGE with 5–20% or 10–20% acrylamide gradient gels (ATTO Corporation), transferred to nitrocellulose membranes (GE Healthcare), blocked with Block Ace (DS Pharma Biomedical), and incubated with rabbit anti-HMGB1 polyclonal antibody (18256; Abcam) or mouse anti-desHMGB1 monoclonal antibody at 4 °C overnight. Next, the membranes were washed with Tris-buffered saline containing 0.02% Tween-20, and then incubated with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare) followed by detection with the Immobilon Western Chemiluminescent detection system (Merck Millipore).