Myd88 antibody

Protein lysates were prepared from purified mouse B cells with RIPA lysis buffer supplemented with protease inhibitor cocktail (Sigma). The protein expression levels of MyD88 and NF‐κB p65 protein were detected by Western blotting. Briefly, equal amounts of protein were electrophoresed on an SDS‐PAGE then transferred onto a nitrocellulose membrane, which was blocked in Tris‐buffered saline with Tween (TBST) containing 5% non‐fat dry milk. Then, the membranes were incubated with rabbit polyclonal MyD88 antibody (Abcam), rabbit monoclonal NF‐κB p65 antibody (Abcam) and rabbit polyclonal β‐actin antibody (Abcam), respectively. Blots were visualized after incubation with goat horseradish peroxidase (HRP)‐conjugated IgG antibodies. Signals were detected using enhanced chemiluminescence (Bio‐Rad).

Left kidneys were dissected from mice 1 day and 28 day after I/R and fixed in 10% formalin. Formalin-fixed kidneys were embedded in paraffin, and 4 μm sections were stained with Hematoxylin and Eosin (H&E). Tubular necrosis was semi-quantitatively analyzed according to previously reported methodology47 (link). Renal MPO, fibronectin, collagen IV and α-SMA was evaluated by immunohistochemistry as the method previously described40 (link). Masson’s trichrome staining was used to evaluate renal fibrosis. Twenty-four hours after I/R, cell apoptosis in paraffin sections was analyzed by TUNEL assay with the in Situ Cell Death Detection kit, POD (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Histopathology and immunohistochemistry scoring was performed by pathologists blinded to the treatment groups. Frozen (day 1) and formalin-fixed (day 28) kidneys were used to perform immunofluorescence analysis with a MyD88 antibody (Abcam) at 4 °C overnight. A Cy3-conjugated secondary antibody was then added at 37 °C for 50 min. Cell nuclei were stained with DAPI.