Vector elite kit

Lungs were fixed in 4% paraformaldehyde, embedded in paraffin, and 5 μm sections were obtained. Sections were dehydrated, deparaffinized, and antigen retrieval was performed using the citrate buffer method [15 (link),16 (link)]. RAGE immunofluorescence was completed using goat polyclonal IgG (AF1145, 1:500, R&D Systems, Minneapolis, MN). Sections were blocked in 5% donkey serum in PBS for 2 hours at room temperature, followed by incubation with primary antibodies at 4°C overnight. Control sections were incubated in blocking serum alone. After overnight incubation, all sections (including the controls) were washed using PBS/triton prior to the application of Alexa Fluor® 488 Rabbit Anti-Goat IgG (Invitrogen, Carlsbad, CA) secondary antibodies for 1 hour at room temperature. For immunohistochemistry, slides were blocked, incubated with primary and appropriate secondary antibodies that utilize HRP conjugation with the Vector Elite Kit (Vector Laboratories; Burlingame, CA). Antibodies included collagen IV (1:500, Abcam, Cambridge, MA, ab6586) and MMP-9 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, sc-6840). The TdT-FragEL DNA Fragmentation Detection Kit (Calbiochem, Rockland, MA) was used to immunohistochemically evaluate apoptosis. No staining was observed in sections without primary or secondary antibody.

We assessed neuronal activation in the fixed brains on the basis of positive Fos-immunoreactivity, seen as a black deposit in the nucleus. Briefly, a one-in-four series of forebrain sections from each animal was incubated in primary Fos antibody (overnight; 4 °C; 1:10 000; rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA), then in secondary antibody (90 min; room temperature; 1:200; biotinylated anti-rabbit; Vector Laboratories, Burlingame, CA, USA) and in an avidin-biotin horseradish peroxidase (HRP) complex (1 hr; Vector Elite kit, Vector). The sections were then incubated in nickel diaminobenzidine to visualize the HRP activity, seen as a black nuclear deposit. The reaction was terminated once an optimal contrast between specific cellular and non-specific background labelling was reached. Sections from each treatment group were processed simultaneously. Sections were mounted on chrome-alum-coated slides, dehydrated in a series of alcohols, cleared in Histoclear and coverslipped.

Brain sections were prepared as described above. The free-floating sections were washed 6 times in PBS and then incubated in 10% normal goat serum at room temperature for 2 hours. The sections were then incubated in the primary rabbit anti-SRC-1 serum (1:2000; catalog no. 2191, Cell Signaling, Beverly, MA) at room temperature overnight, followed by biotinylated anti-rabbit secondary antibody (1:1000; Vector Laboratories, Burlingame, CA) for 2 hours. Next, the sections were incubated in the avidin-biotin complex (Vector Elite kit; 1:500) and then in 0.04% diaminobenzidine and 0.01% hydrogen peroxide. After dehydration through graded ethanol, the slides were immersed in xylene and coverslipped. Images were analyzed using a Leica 5500 microscope configured with a bright-field camera (Leica, Heidelberg, Germany).

We assessed neuronal activation on the basis of positive Fos‐immunoreactivity, seen as a black deposit in the nucleus as previously described (Spencer et al. 2004a,b; Mouihate et al. 2010) (n = 8 AD and nine CR nonstressed, n = 9 AD and eight CR stressed). A one‐in‐four series of forebrain sections from each animal was incubated in primary Fos antibody (O/N; 1:10,000; rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA), then in secondary antibody (1.5 h; 1:500; biotinylated anti‐rabbit; Vector Laboratories, Burlingame, CA) and in an avidin‐biotin horseradish peroxidase (HRP) complex (ABC; 45 min; Vector Elite kit; Vector). The sections were then incubated in nickel diaminobenzidine (DAB) to visualize the HRP activity, seen as a black nuclear deposit. The reactions were terminated once an optimal contrast between specific cellular and nonspecific background labeling was reached. Sections from each treatment group were processed simultaneously. Sections were mounted on polylysine‐coated slides, dehydrated in a series of alcohols, cleared in Xylene, and coverslipped.

The specimens were oriented for the transverse fibers sectioning. For each P. major muscle, 10 serial cross-sections (10 μm thick) were cut on a cryostat microtome at −20°C and mounted on poly-L-lysine coated glass slides (Sigma-Aldrich, St. Louis, MO, United States). For immunohistochemistry, the avidin-biotin-peroxidase complex (ABC) method was used. Briefly, the sections were rinsed in phosphate buffer saline and incubated in 5% normal goat serum (for 30 min at room temperature) to reduce the non-specific binding of the secondary antibodies. The sections were then incubated at 4°C in a humid chamber for 24 h with the monoclonal mouse antiserum anti-VIM and the polyclonal rabbit antiserum anti-DES (61013 and 10570, Progen Biotechnik GmbH, Heidelberg, Germany, respectively) both diluted 1:1000. After washing, the sections were incubated at room temperature for 1 h with the biotin-conjugated goat anti-mouse IgG and biotin-conjugated goat anti-rabbit IgG secondary antibodies, both diluted 1:200 (Vector Laboratories, Burlingame, CA, United States), and then treated with ABC (Vector elite kit, Vector Laboratories, Burlingame, CA, United States). The immune reactions were visualized through a 3,3′-diaminobenzidine (DAB) chromogen solution (Vector DAB kit, Vector Laboratories, Burlingame, CA, United States).