Rabbit anti hsp25

Primary antibodies (supplier; dilutions) used in this study were as follows: rabbit anti-Calbindin (Swant; 1∶10,000); goat anti-ChAT (Millipore; 1∶500); rabbit anti-GFAP (DAKO; 1∶8,000); mouse anti-GM130 (BD Transduction; 1∶100); rabbit anti-Hsp25 (Enzo; 1∶8,000); rabbit anti-Iba-1 (Wako; 1∶5,000); rat anti-Ki67 (DAKO; 1∶200); rat anti-Mac2 (Cedarlane;1∶2,000); mouse anti-NeuN (Millipore; 1∶1,000); rabbit anti-p53 (Leica; 1∶1,000). For avidin– biotin–peroxidase immunocytochemistry biotinylated secondary antibodies from Vector Laboratories, diluted 1∶200 were used. FITC-, Cy3-, and Cy5-conjugated secondary antibodies raised in donkey (Jackson ImmunoResearch) diluted at 1∶200 were used for confocal immunofluorescence.

Western blot analysis was carried out on mouse embryonic fibroblast cell lines derived from HSPB1^+/+ and HSPB1^−/− mice and grown in standard DMEM media supplemented with 10% fetal calf serum and 1X nonessential amino acids. Subconfluent cells were treated with 200 ng/ml lipopolysaccharide (LPS, E. coli O55:B5; SigmaAldrich) and cell extracts prepared from cells after 0.5 to 6 hours exposure. Twenty micrograms of protein was fractionated by SDS gel electrophoresis and transferred to PDVF membrane by standard techniques following by probing with the following primary antibodies: rabbit anti-IκB-α, Cell Signaling, Danvers, MA), rabbit anti-phospho-NF-κB p65 (Ser536) (Cell Signaling), mouse anti-Lamin A (Santa Cruz Biotechnology, Dallas, TX) and rabbit anti-HSP25 (Enzo Life Sciences, Farmingdale, NY). Secondary antibodies were horseradish perioxidase coupled goat anti-mouse or anti-rabbit (Jackson ImmunoResearch, West Grove, PA). Chemilumenscence (SuperSignal West Femto kit; Thermo Scientific, Norcross, GA) was captured by X-ray film.