Anti rabbit hrp conjugated antibody

Hippocampal slice cultures (400 μm thick) were prepared from 7‐day‐old C57BL/6J mice as described.28 After 3 days, culture medium was changed to a hypoxic/hypoglycemic medium (75% Neurobasal, 25% HBSS, 1% L‐glutamine, bubbled with 99.4% nitrogen). Slices were placed in a hypoxia incubation chamber (Stemcell Technologies, Vancouver), and nitrogen gas was flowed into the chamber, followed by incubation at 37^o for 1 h. The culture medium was replaced by normal culture medium containing glucose, and slices were placed in a tissue culture incubator. After 2 h of culture, 1 μmol/L of PAP‐1 or 10 μmol/L of minocycline were added and slices cultured for 3 days. Slices were fixed in 4% formaldehyde and stained with anti‐NeuN (1:400; Chemicon) and anti‐Iba1 (1:400; Wako Chemical) followed by secondary Alexa‐Fluor®488‐conjugated anti‐mouse or Alexa‐Fluor®568‐conjugated anti‐rabbit antibodies (1:700; Life Technologies). For Western blotting, slices were lysed and protein electrophoresed and visualized as previously described.14 The following primary antibodies were used as follows: anti‐PSD95 (1:1000) and anti‐GAPDH (1: 3000; both Cell Signaling Technology). The secondary antibody was an HRP‐conjugated anti‐rabbit antibody (1:3000; Amersham Biosciences).

Keratinocytes were lysed in protein extraction buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1% Nonidet P-40, 4 mM EDTA) containing proteinase inhibitors cocktail (Complete Mini, EDTA-free; Roche Diagnostics, Mannheim, Germany). Lysates were incubated for 30 min on ice and centrifuged at 15,000 × g for 30 min at 4°C. Supernatants were collected and protein concentrations were measured using the Bradford assay (BioRad, Hercules, CA). For each sample, 40 μg of total protein was resolved on NuPAGE Novex 3%–8% Tris-Acetate gel electrophoresis (Invitrogen, Carlsbad, CA) and electrotransferred to nitrocellulose membranes (Invitrogen, Carlsbad, CA). For type VII collagen analysis, blots were probed with a monospecific polyclonal anti C7 antibody (a generous gift from Dr A. Nystrom). Antibodies against vinculin (Abcam) or α-tubulin (Sigma, St. Louis, MO) were used as loading controls. Visualization was performed by incubating with HRP-conjugated anti-rabbit antibody (Amersham, Burlington, MA) and West Pico Chemiluminescent Substrate (Pierce, Rockford, IL).