Anti c fos antibody

Cytoplasmic and nuclear lysates were prepared as previously described [24 (link)]. To prepare total cell lysates from granulocytes, the cells were precipitated in 10% trichloroacetic acid (Wako) for 30 min on ice. The TCA-precipitated fraction was treated with a lysis solution containing 9 M urea, 2% Triton X-100, and 1% dithiothreitol, and was disrupted by ultrasonication, followed by an addition of 2% lithium dodecyl sulfate and further ultrasonication. The proteins were separated on a 10% polyacrylamide gel, transferred onto Immobilon-P Transfer Membranes (Millipore, Billerica, MA, USA), and blotted with rabbit polyclonal anti-c-Fos antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit polyclonal anti-actin (Sigma), followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz). Signals were detected by ECL Western Blotting Detection Reagents (GE Healthcare, Buckinghamshire, UK).

For VIP immunostaining, brains of mixed-sex PN0 pups were harvested at 2 h after light onset, immersion-fixed in 4% paraformaldehyde at 4°C for 6 h, cryoprotected in 30% sucrose, coronally sectioned at 20-μm thickness using a cryostat, and thaw-mounted onto gelatin-coated slides. For cFos immunostaining, adult brains described above were cryosectioned at 50-μm thickness, and floating sections were collected into phosphate-buffered saline (PBS). IHC for VIP was performed using an anti-VIP antibody [1:1000; a gift from Dr. Dick Swaab (21 –23 (link))], and IHC for cFos was performed using an anti-cFos antibody [1:400; Santa Cruz Biotechnology, Dallas, TX, USA (24 (link)–26 (link))]. Sections were incubated in the primary antibody for 1 week (for VIP IHC) or 48 h (for cFos IHC). The sections were then incubated sequentially with a secondary biotinylated donkey anti-rabbit antibody, avidin–biotin complex (ABC; Vector Labs, Burlingame, CA, USA), and reacted with diaminobenzidine with (cFos IHC) or without (VIP IHC) 0.05% nickel enhancement for color detection. After the color reaction, floating sections were washed and mounted onto gelatin-coated slides. All slides were counterstained with 1% methyl green, a nuclear stain, to visualize the perimeters of the SCN before dehydration and coverslipping.

RANKL was purchased from Peprotech (London, UK). Alpha-minimum essential media (α-MEM), fetal bovine serum (FBS), penicillin/streptomycin (P/S) and Dulbecco’s phosphate buffered saline (DPBS) were obtained from Gibco (Gaithersburg, NY, USA). TRAP assay kit was obtained from Sigma Aldrich (Saint Louis, MO, USA). Osteo assay surface multiple well plates were obtained from Corning, Inc. (New York, NY, USA). Anti-c-Fos antibody, anti-TRAF6 antibody and anti-β-actin antibody were obtained from Santa Cruz (CA, USA). Anti-NFATc1 antibody was purchased from BD Pharmingen (San Diego, CA, USA). Anti-MMP-9 antibody and anti-CTK antibody were purchased from Abcam (Cambridge, MA, USA). Anti-total-ERK antibody, anti-phospho ERK antibody, Anti-total-JNK antibody, anti-phospho JNK antibody, Anti-total-p38 antibody and anti-phospho p38 antibody were purchased from Cell signaling (Beverly, MA, USA). Anti-NFATc1 antibody was purchased from BD Pharmingen (San Diego, CA, USA).PCR primers were obtained from Genotech (Daejeon, Korea). All of the chemicals used in the experiments were of analytical grade or complied with the level required for cell culture.

The rats were perfused with phosphate buffered saline (PBS) and paraformaldehyde after ischemia-reperfusion, followed by the collection and postfixation of the brains. Subsequently, we transferred the specimens to 0.1 M phosphate buffer containing 30% sucrose and kept them at 4°C for 72 hours. Twenty-five-micrometer sections through the DMV were cut in a frozen section machine. The sections were washed twice with PBS, then blocked with 3% BSA for 2 hours, and incubated with anti-c-Fos antibody (dilution 1 : 100, Santa Cruz, USA) overnight at 4°C. Repeat the washing with PBS for three times. After that, incubate sections with a second antibody at room temperature for 2 hours. The samples were then observed under an immunofluorescent microscope.

The brains were prepared using the same method as in histology and cut into 40-µm-thick coronal sections with a cryotome (CM300, Leica). The sectioned brain regions encompassed the habenula and the RMTg. Brain sections were incubated in primary antibodies for 1 h at 37 °C. The primary antibodies were diluted in PBS containing 3% bovine serum albumin (BSA) and 0.2% Triton X-100. The following primary antibodies were used: anti-c-Fos antibody (1:500; #SC-52G, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and anti-GABA antibody (1:500; #A2052, Sigma-Aldrich, St. Louis, MO, USA). Next, the sections were washed with PBS and incubated in a cocktail of Alexa Fluor® 488 donkey anti-goat (1:500; #A-11055, Thermo Fisher Scientific) or Alexa Fluor® 647 donkey anti-rabbit (1:500; #711-605-152, Jackson Immuno Research Inc., West Grove, PA, USA) conjugated secondary antibodies in PBS containing 3% BSA and 0.2% Triton X-100 for 2 h at room temperature. The secondary antibody was washed with PBS and further incubated with Hoechst (1:1000; #H3570, Thermo Fisher Scientific) at room temperature for 10 min. The sections were immersed in mounting solution and images were captured using a TCS SP8 dichroic/CS microscope (Leica).

Thirty min after being subjected to the TST, the mouse was anesthetized as above. After confirming that the mouse was deeply anesthetized, the brain was fixed by transcardiac perfusion with 4% paraformaldehyde in PBS. The brains were sliced coronally (100 μm in thickness) using a microslicer (PRO7, Dosaka, Kyoto, Japan). The free-floating slices were reacted with an anti-c-Fos antibody (1: 200, goat, Santa Cruz, Dallas, TX) dissolved in PBS containing 0.3% BSA and 0.3% Triton-X-100, at 4°C for 48 h. The immunoreaction was visualized using a secondary antibody (rabbit anti-goat IgG, Vectastain Elite ABC-HRP Kit, Vector Labs, Burlingame, CA) and 3,3'-Diaminobenzidine (DAB; Tokyo Chemical Industry, Tokyo, Japan) at room temperature in accordance with the manufacturer's instructions. For statistical analysis, the number of c-Fos-immunoreactive cells was quantified using Image-J software. Briefly, fields (0.16 mm²) of c-Fos immunoreactivity were acquired using a microscope using a CCD camera. DAB-positive nuclei, of which the diameters were longer than 7 μm, were considered as anti-c-Fos-immunoreactive neurons. The immunoreactive cell numbers were normalized to cells/mm².

Total proteins were extracted using Cell Lysis Buffer purchased from Cell Signaling Technology (Beverly, MA). The extracted proteins were separated by 10% SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes. The membranes were then probed with anti-phospho-ERK5 and anti-ERK5 antibodies that were purchased from Cell Signaling Technology, anti-c-Fos antibody from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), and anti-β-Actin pAb-HRP-DirecT from MBL, Nagano. Primary antibodies were detected using horseradish peroxidase-conjugated secondary antibodies and visualized using LumiGLO Reagent and Peroxidet purchased from Cell Signaling Technology.