Rabbit anti caveolin 1

For co- localization experiments, after peptide incubation, cells were first rinsed twice with PBS to remove non-internalised peptide and fixed with paraformaldehyde 4% for 20 min. The cells were washed for 10 min in 10 mM PBS/20 mM glycine, permeabilised with 10 mM PBS/20 mM glycine containing 0.005% saponin for 7 min, and blocked with PBS/20 mM glycine 1% albumin. αvβ3 integrin was performed by using mouse anti-human integrin αvβ3 primary antibodies (Chemicon) and Alexa-fluor 568 goat anti-mouse secondary antibodies (Molecular Probes, Invitrogen). Endocytic vesicles were localised by incubating samples first with mouse anti-clathrin monoclonal (ABR) and rabbit anti-caveolin 1 (Abcam) primary antibodies and then with Alexa-fluor 568 goat anti-mouse and anti-rabbit secondary antibodies (Molecular Probes, Invitrogen), respectively. Lysosomes were localised with rabbit anti-LAMP 2 polyclonal (Abcam) primary antibodies and with 568 goat anti-rabbit secondary antibodies (Molecular Probes, Invitrogen).
All samples were then observed with a confocal microscope with a 63× oil immersion objective. Co-compartimentalisation between the peptide and the endocytic markers was analysed with ImageJ analysis software plugin.

DMEM, fetal bovine serum, penicillin/streptomycin, trypsin / EDTA, PrestoBlue Cell Viability Assay and Bodipy-Lactosylceramide-BSA were purchased from Life Technology. DCFDA cellular reactive oxygen species detection assay kit was purchased from Abcam. The Protein Carbonyl Assay kit was from Cayman Chemical Company. ECL Prime Western Blot detection kit was from GE Healthcare. Rabbit anti-caveolin 1 and anti-cavin 1 were purchased from Abcam. Anti-p38 α and anti-calnexin antibodies were from Santacruz Biotechnology. Anti-golgin antibodies were from Molecular probes and mouse anti-Ubiquitin antibodies from Calbiochem. Endocytosis specific inhibitors were purchased from Merck. The caveolin 1 specific siRNA, the negative control, lipofectamine and Opti-MEM were purchased from Invitrogen. Alexa-fluor conjugated secondary antibodies were from Invitrogen also. All other chemicals were from Sigma.

The animals were sacrificed at 0, 4, 26, and 50 h after reperfusion with or without HPC (n = 3 in each group), respectively. Rats were killed under chloral hydrate anesthesia. The brain tissue was incised into 2-mm coronal slices with a brain matrix, and the CA1 regions of bilateral hippocampi were quickly divided under the stereomicroscope. The protein extraction and western blotting procedure were performed as described previously^{63 (link)}. In order to detect the level of Atp1b1, membranous and cytosol protein were extracted with a membrane and cytosol protein extraction kit (Beyotime Biotechnology). The primary antibodies included rabbit anti-Ngb (diluted 1:200; Proteintech Group), mouse anti-Atp1b1 (diluted 1:5000; Millipore), mouse anti-GAPDH (diluted 1:10,000; Proteintech Group), and rabbit anti-Caveolin-1 (diluted 1:2000; Abcam, Inc., Cambridge, UK). Densitometric analysis for the quantification of the bands was performed with the image analysis software (Quantity One, Bio-Rad Laboratories, Inc., Hercules, CA). Relative optical densities of protein bands were calibrated with GAPDH or Caveolin-1 and normalized to those in the Sham-operated rats.