Biotinylated goat anti rabbit igg

Mice were sacrificed by CO2 suffocation. Tumors were extracted and washed using normal saline solution. The samples were then instantly frozen in liquid nitrogen and sectioned. All tissues were stained with hematoxylin and eosin for the histologic evaluation. Light microscopy images were acquired with an Olympus IX51 microscope.
Immunohistochemical analysis was performed on xenografts and GEMM tissue samples as described previously (14 (link), 21 (link)). Briefly, unstained 5-µm sections were fixed by standard techniques before antigen retrieval in ethylenediaminetetraacetic acid buffer. The samples were then incubated by the 1:50 dilution of antiperiostin rabbit polyclonal IgG (ab14041; Abcam) overnight at 4°C. The sections were washed in Tris-buffered saline and polysorbate 20 mixture and incubated with biotinylated goat antirabbit IgG (Abcam) for 1 h. The staining was developed by incubation in liquid 3,3′-diaminobenzidine (Dako) for 1 min. Slides were washed in distillated water and counterstained with hematoxylin (Richard-Allan Scientific) and dehydrated before mounting. Two esophageal cancer tissue arrays (ES2081 and ES2001; US Biomax) were stained for periostin expression with slight modification of the technique described above. The scoring for periostin expression was done as previously described (13 (link)).

Following our previously reported methods,^{(28 )} femurs were cleaned of adherent tissue, fixed overnight at 4 °C in 4% paraformaldehyde in PBS, rinsed in PBS, dehydrated through an ethanol series, cleared in xylene, embedded in paraffin, and cut into 5-μm sections. Deparaffinized and rehydrated sections were incubated with 3% hydrogen peroxide in methanol to block endogenous peroxidase and with protein blocker (Abcam, Cambridge, MA, USA) to block the nonspecific binding of antibodies. The sections were then reacted with rabbit IGF1R antibody (1:200) (Aviva Systems Biology, San Diego, CA, USA) at 4 °C overnight. After washing with PBS, the sections were incubated with biotinylated goat anti-rabbit IgG (Abcam), then streptavidin peroxidase (Abcam), and visualized by 3,3′-diaminobenzidine.

Immunohistochemistry was carried out as described previously (Zan et al. 2014 (link)). After being blocked, coronal sections (n=6 rats/group) were incubated with rabbit anti-MAP2 (a mature neuronal marker, 1:1000, Abcam) at 4°C overnight and then with biotinylated goat anti-rabbit IgG (1:1000, Abcam) for 1 h at room temperature. The immunoreactions were visualized with diaminobenzidine-H₂O₂ solution. Control sections were processed without the primary antibody and showed no positive signals. MAP2-positive cells were counted around the lesion under a 40× objective from 15 randomly selected locations per rat (5 fields per section × 3 sections per rat). Quantifications were averaged and expressed as positive cells per field. Sections were selected and analyzed by an investigator blinded to the experimental cohort.