Abc staining kit

All animal experiments were performed in accordance with the guidelines of MD Anderson Cancer Center’s Institutional Animal Care and Use Committee. C57BL/6 mice (6- to 8-weeks old; from the National Cancer Institute) were hydrodynamically transfected [31 (link)] with 100 μg of plasmid (pEF4-myc or pEF4-CD74-myc [4 (link)]; kindly provided by Dr. Idit Shachar, The Weisman Institute of Science, Rehovot, Israel). Mice were challenged with agonistic anti-Fas antibody Jo2 (0.4 μg/g; BD Biosciences) 24 h post transfection and monitored for survival up to 6 h post challenge. Livers were harvested at the time of death/sacrifice, paraffin embedded, and cut to 5 μm sections.
Formalin-fixed, paraffin-embedded liver tissue sections on microscope slides were treated with an unmasking kit (1:500; Vector Laboratories). Sections were then stained with hematoxylin and eosin (H&E; both from Fisher Scientific). Immunohistochemical staining was performed using a mouse monoclonal anti-myc antibody (1:500; Sigma-Aldrich) and a rabbit polyclonal anti-cleaved caspase-3 antibody (1:500; Cell Signaling), followed by staining and developing with an ABC staining kit (Vector Laboratories), according to manufacturer’s protocol. Stained samples were viewed using a Zeiss Axioskop 2 plus microscope (Carl Zeiss) with 4× and 20× magnification objectives.

The procedure for immunohistochemical analysis has been described elsewhere^{60 (link),93 (link)}. Briefly, blind-coded sagittal sections were incubated with the following primary antibodies; anti-α-synuclein(Syn1, #610787, BD bioscience, San Diego, CA, 1:250), anti-α-synuclein (Syn211, # 36-008), anti-NeuN(#MAB377, 1:1000), anti-GFAP (#MAB3402, 1:1000), anti-TH (# AB152, Millipore, County Cork, Ireland, 1:1000), and anti-Iba1 (#019-19741, Wako, Richmond, VA, 1:1000). To detect protease K (PK) resistant α-synuclein aggregates, the sections were pretreated with PK (10 µg/ml) for 8 minutes prior to incubating with anti-α-synuclein antibody^{94 (link)}. After overnight incubation at 4 °C, the sections were incubated with biotinylated secondary antibodies and subsequently detected utilizing an ABC staining kit (both from Vector Laboratories, Burlingame, PA). All sections were imaged by an Olympus BX41 microscope, and the immunoreactivity levels were determined by utilizing ImageJ (NIH). To determine α-synuclein pathology, neurodegeneration, microgliosis, and astrogliosis, the optical density of α-synuclein and GFAP per field (230 mm×184 mm) and the numbers of α-synuclein-positive, NeuN-positive, TH-positive fibers, Iba1-positive cells per indicated field were analyzed using Image Quant 1.43 program (NIH).

Lgr4 was PCR-amplified from mouse lungs using the following primers: mLgr4 FOR: 5′-TCTTGTTCATCACTGCCTGC-3′, REV: 5′-AGCTGTCCGAGACAAAGGAA-3′. Amplified cDNAs were cloned into the vector pGEM-T easy (Promega) and linearized with the appropriate restriction enzymes. Probe preparation and in situ protocol were performed as previously (44 (link)). When colabeling was desired, after in situ, the sections were incubated with primary antibodies (anti-GnRH, 20075, Immunostar) diluted 1:1000 in PBS–Triton 0.1%, overnight at room temperature (RT) (45 (link)). After 3 washes with PBS–Triton 0.1%, the slides were incubated for 2 hours at RT with biotin-conjugated goat secondary antibodies (Vector Laboratories), diluted 1:300 in PBS and, after further washes, with the avidin–biotin complex (ABC staining kit, Vector Laboratories). The sections were reacted with 3,3′-diaminobenzidine (DAB, Vector Laboratories) and mounted in an aqueous compound formed by PBS and glycerol (3:1). Images were acquired using a Leica DM5500B microscope (Leica), equipped with a DCF295 camera (Leica) and DCViewer software (Leica), and then processed with Abode Photoshop CS6 and Adobe Illustrator CS6 software.