Vector vip peroxidase hrp substrate kit

Immunohistochemical detection of transported CTB and FG was carried out on coronal sections. Sections were incubated with a primary antibody against CTB raised in goat (1:2000; List Biologicals, INC, 703) and rabbit antibody Anti-Fluorescent Gold (1:2000; Chemicon, AB153), diluted in a solution containing 5% of normal donkey serum (NDS) (Jackson inmunoresearch Laboratories, 017-000-121), 5% normal swine serum (Jackson immunoresearch Laboratories, 014-00-121), 0.04% triton X-100 in phosphate buffer (PBS) pH 7.4 overnight. After rinsing in PBS, sections were incubated for 2 h in a solution containing 5% of NSwS, 5% NDS, swine anti-rabbit IgG (1:50, Dako, Z0196) and donkey anti-goat (Jackson Immunoresearch, 705-065-147) diluted in PBS for 90 min; after washes sections were incubated in a solution containing 5% NDS and Goat PAP (1:600 Sigma, P1901) and afterwards washed in PBS and visualized in brown with DAB (Sigma, D5637). Section were washed in PBS and incubated in a solution containing 5% NSwS and Rabbit PAP (1:50 Dako, Z0113 diluted in PBS and visualized using Vector VIP Peroxidase (HRP) Substrate Kit (Vector laboratories, SK-4600). Sections were mounted on gelatin-coated glass slides, dried at RT and coverslip with Dpex (VWR International).

IHC procedures were completed as previously described [36 (link)]. For antigen retrieval, slides were incubated in 1x citrate buffer (pH 6.0, DBS, Pleasanton, CA) at 100°C in a water bath for 10 min. After peroxidase and protein blocking and treatments, the tissue sections were incubated with the anti-human c-Met antibody (Ab) (2μg/mL, rabbit polyclonal Ab, Santa Cluz Biotechnology, Dallas, TX) at room temperature in a humid chamber for 1 hr. The sections were then visualized using the CSAII kit (Dako, Carpinteria, CA) and Vector VIP Peroxidase (HRP) Substrate Kit (Vector Laboratories, Burlingame, CA) according to the manufacturers' instructions. The sections were counterstained with hematoxylin. Photographs of the IHC staining were taken for analysis using the Nikon Eclipse Ti microscope and NIS elements software (Nikon, Melville, NY). IHC staining intensity values of specimens on slides without the primary Ab were subtracted from the corresponding specimen with the primary Ab to exclude the influence of pigmentation and background staining.

Immunohistochemistry was performed on free-floating sections (100-μm thick). Sections were washed in phosphate-buffered saline (PBS) and incubated for 30 min with 5% diluted normal donkey serum (Jackson ImmunoResearch Europe, Ltd., Suffolk, UK) and 0.5% Triton X-100; then incubated overnight at room temperature in the same solution with the primary antibody goat-anti GFP (#ab6673, 1:500; Abcam, Cambridge, UK). Secondary amplification and detection were performed using the VectaStain ABC Elite Kit (#PK-6101; Vector Labs, Eurobio, Courtaboeuf, France) and the Vector VIP Peroxidase (HRP) Substrate Kit (#SK-4600) that were used according to the manufacturer recommendation. Finally, sections were rinsed in tap water and mounted. Using the Fiji software, images were converted to 8-bit format, then thresholded, and a region of interest (ROI) was drawn on the image according to the brain structures in the Allen Mouse Brain Atlas (2004). The number of particles in the ROI was then counted with the Fiji particle analyzing tool. All slides were analyzed with the experimenter blind for the treatment. Six animals were counted for control condition and 6 for FUS-treated condition. Several slices were counted for one animal.