Horseradish peroxidase conjugated goat anti mouse antibody

Six rats from each group were euthanized 48 hours post-occlusion. The brains were carefully removed and placed in chilled saline. Total protein was isolated directly from the ischemic penumbra after cell lysis and then added to Laemmli buffer (75 mM Tris-HCl containing 2% sodium dodecyl sulfate (SDS), 10% glycerol, 2% 2-mercaptoethanol, 0.002% bromphenol blue). The samples were heated to 95°C for 10 minutes and then separated on 10% Tris/glycine/SDS acrylamide gels (Bio-Rad, Hercules, CA, USA). The proteins were subsequently transferred to polyvinylidene difluoride (Minipore, Billerica, MA, USA) membranes and blocked for 2 hours at room temperature in 5% non-fat milk. The immunoblots were incubated for 2 hours at 37°C with mouse anti-occludin or mouse anti-ZO-1, both with β-actin monoclonal antibody (serving as the control) (all at 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing (3 × with Tris-buffered saline/0.05% Tween-20), the blots were incubated with horseradish peroxidase-conjugated goat anti-mouse antibody (1:1,000; Santa Cruz Biotechnology) for 1 hour at 37°C. The membranes were then stained with tetrazotized o-dianisidine and β-naphthyl acid phosphate and analyzed with an electrophoresis gel imaging system (Bio-Rad) to obtain OD values. The levels of occludin and ZO-1 were then expressed as percentages relative to β-actin.

Western blotting was performed, as described previously (17 (link),18 (link)). Briefly, the total protein was isolated from the harvested cells and the quantity of protein was measured using a BCA protein assay. Samples containing 25 µg protein were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Inc. Hercules, CA, USA). Non-specific binding sites were blocked for 1 h with 5% non-fat milk, prior to an incubation with the following primary antibodies diluted in PBS at 4°C overnight: Anti-CDK4 (1:1,000), anti-cyclin D1 (1:1,000), anti-p21^Cip1 (1:1,000), anti-p27^Kip1 (1:1,000), anti-cytochrome c (1:1,000), anti-caspase-9 (1:1,000), anti-caspase-3 (1:1,000), anti-PARP (1:1,000), anti-c-PARP (1:1,000), anti-Bcl-2 (1:1,000), anti-Bax (1:1,000), and anti-β-actin (1:1,000) Subsequently, a secondary horseradish peroxidase-conjugated goat anti-mouse antibody (1:1,000; Santa Cruz Biotechnology, Inc.) was added for 1 h at room temperature. The blots were visualized using an enhanced chemiluminescence system (Amersham, Buckinghamshire, UK), and the density of β-actin served as an internal loading control.

IECs were isolated from the jejunum, protein extracts were prepared, and then immunoblotting was performed as described previously.19 (link), 38 (link) Protein levels were expressed as a ratio to glyceraldehyde-3-phosphate dehydrogenase protein levels except for analysis of Egfr recombination in IECs of IEC-EgfrKO mice, in which β-actin was used as a loading control. In some experiments, IECs were separated further into nuclear and cytosolic fractions for immunoblotting as described previously.39 (link), 40 (link) For cytoplasmic extracts, protein levels were expressed as a ratio to glyceraldehyde-3-phosphate dehydrogenase protein levels, whereas for nuclear extracts, protein levels were expressed as a ratio to histone H3 protein levels. Western blotting was performed using primary antibodies outlined in Table 2. Secondary antibodies were the corresponding horseradish-peroxidase–conjugated goat anti-mouse antibody or goat anti-rabbit antibodies (Santa Cruz Biotechnology, Santa Cruz, CA).

Small intestinal extracts were prepared, subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis using a 4%–20% gradient (Tris-Glycine Midi Gel; Invitrogen), and analyzed by immunoblotting.⁸ (link) Blots were stripped before re-probing with antibodies to the different bile acid transport proteins or re-probing with antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to normalize for protein load. Protein expression was quantified by densitometry using a Microtek (Hsinchu, Tawain) ScanMaker i900 and FujiFilm (Tokyo, Japan) Multiguage 3 software, and expression data were normalized to levels of the GAPDH loading control. Sources of the antibodies used in the study were as follows: anti-mouse Asbt,⁷ (link) anti-mouse Ostα and anti-mouse Ostβ,⁸ (link) anti-mouse Ibabp (anti–fatty acid binding protein 6, catalog no. ab91184, lot GR245572-3; Abcam), anti-GAPDH (catalog no. MA5-15738; Thermo Fisher Scientific), horseradish-peroxidase–conjugated goat anti-rabbit antibody (catalog no. A9169, lot 015M48581; Sigma-Aldrich), and horseradish-peroxidase–conjugated goat anti-mouse antibody (catalog no. sc-2005, lot K1915; Santa Cruz Biotechnology, Dallas, TX).