Ecl plus chemiluminescence kit

If not otherwise specified, chemicals and reagents were purchased from Sigma (Steinheim, Germany) or Roth (Karlsruhe, Germany). Pefabloc was obtained from Roche (Mannheim, Germany) and staurosporine was purchased from Enzo (Lausen, Switzerland). Ultrapure low-melting-point agarose, slice culture media, supplements and heat inactivated horse serum were obtained from Invitrogen (Darmstadt, Germany). The bicinchoninic acid assay kit was purchased from Pierce (Thermo Scientific, Rockford, USA). The ECL plus chemiluminescence kit was purchased from GE Healthcare (Buckinghamshire, UK). Monoclonal anti-PrP antibody 4H11 has been described previously (Ertmer et al., 2004 (link)). The antibody is directed against the globular domain of the protein. Anti-actin antibody was obtained from MP Biomedicals (Eschwede, Germany) and rabbit polyclonal anti-β-3-tubulin antibody was purchased from Covance HISS Diagnostics (Freiburg, Germany). Anti-calbindin D-28K antibody was purchased from Swant (Marly, Switzerland), anti-GFAP antibody was obtained from Dako Cytomation (Hamburg, Germany), anti-iba-1 antibody was purchased from Wako Pure Chemicals Industries (Osaka, Japan), and anti-lamp-1 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, USA). Fluorescein-conjugated secondary antibodies were purchased from Dianova (Hamburg, Germany) or from Life Technologies (Darmstadt, Germany).

Immuno-dot blot analysis was performed using ApCPEB PLD aliquots at a concentration of 10 μM in PBS at pH 7.0. For EGCG and AmB studies, ApCPEB PLD in the monomeric state was prepared by denaturing it in 6 M Gdn·HCl, considered as the starting time of the reaction. Next, samples were diluted in 5 mM potassium phosphate + 150 mM NaCl, pH 7.4 to a final protein concentration of 2.5–5 μM. DMSO (AmB vehicle) or 4:1 M excess of AmB and EGCG was added. In all the cases, 2 μl of the sample was spotted onto a nitrocellulose membrane. After blocking the membrane for 1 h at room temperature with 10% non-fat milk in TBS containing 0.01% Tween-20, the membrane was incubated at room temperature for 1 h with the polyclonal specific anti-oligomer A11 antibody or the fibril-specific monoclonal antibody OC, diluted to 1:1000 in 3% BSA /TBS-T. The membranes were washed 3 times for 5 min each with TBS-T before incubating at room temperature for 1 h with the anti-Rabbit HRP linked secondary antibody diluted 1:5000 in 3% BSA/TBS-T. After washing the membranes 3 times in TBS-T buffer, the blots were developed with ECL Plus chemiluminescence kit from Amersham-Pharmacia (GE Healthcare).

Samples for protein analysis were prepared and analysed from fermenting, respiring and sporulating cells as published (28 (link)). Note that 25 μg of total protein extract was run on a 4–20% gradient gel (BioRad, USA) for 1 h. Proteins were transferred onto ImmobilonPSQ membranes (Millipore, France) using an electro-blotter system (TE77X; Hoefer, USA) and a modified Towbin buffer (48 mM Tris base, 40 mM glycine and 0.1% SDS) and methanol (20% vol/vol anode; 5% vol/vol cathode) for 2 h. Proteins were detected using a monoclonal anti-myc-horseradish peroxidase antibody (Life Technologies, USA) at 1:1000. The antibodies were incubated in hybridization buffer overnight. The signals were revealed using the ECL-Plus Chemiluminescence kit (GE Healthcare, USA) and the ImageQuant 350 system (GE Healthcare, USA). Band intensities were normalized and quantified using the ImageQuant TL 7.0 software and default parameters. A polyclonal antibody against Pgk1 (Invitrogen, USA) was employed as a loading control.

24 h and 48 h after ischemia retinal tissue for analysis of protein expression was harvested. Total protein from ¾ of retina was extracted and processed for Western Blot as described previously. The membranes were blocked with 5% skim milk in Tween20/PBS and incubated in the recommended dilution of protein specific antibody (p-ERK1/2 #4370, p-p38 #9211, p-JNK #9251, cleaved Caspase-3 #9664, Bax #2722, Bcl-2 #2876, Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. After incubation with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (GE Healthcare, Freiburg, Germany), proteins were visualized using the ECL plus Chemiluminescence Kit (GE Healthcare). For normalization, blots were re-probed with ERK1/2 (#4695), p38 (#9212), JNK (#9258), Caspase-3 (#9665) and ß-Actin (#4967S, all Cell Signaling Technology, Danvers, MA, USA). Relative changes in protein expression in IR injured retinas either with or without ALF-186 were calculated in relation to the corresponding non-ischemic retinae.

24 h after ischemia retinal tissue for analysis of protein expression was harvested. Total protein from ¾ of retina was extracted and processed for Western Blot as described previously. The membranes were blocked with 5% skim milk in Tween20/PBS and incubated in the recommended dilution of protein specific antibody (p-NF-κB #3033, TNF-α #3707S, Cell Signaling Technology, Danvers, MA, USA, Hsp-70 #ab31010, Hsp-90 #ab13492, IL-6 #ab25107, Abcam, Cambridge, UK, sGC-β₁ #160897 Cayman Chemical, Ann Arbor, Michigan, USA) overnight at 4 °C. After incubation with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (GE Healthcare, Freiburg, Germany), proteins were visualized using the ECL plus Chemiluminescence Kit (GE Healthcare). For normalization, blots were re-probed with NF-κB (#8242, Cell Signaling) and ß-Actin (#4967S, Cell Signaling). Relative changes in protein expression in IR injured retinae either with injection of ALF-186 or PBS were calculated in relation to the corresponding non-ischemic retinae.
Densitometric analysis of individual phosphorylation was performed using the Image-J open source software (ImageJ, Version 2.00-rc-44/1.50e, open source image processing software) comparing the relative changes in protein expression in IR injured retinae with each intervention and calculated in relation to the corresponding non-ischemic retinae.