S2438

The floating sections were rinsed in 0.01 M PBS (pH 7.4), treated 30 min in 0.3% H₂O₂ in PBS, and incubated in blocking solution (10% bovine serum in PBS) for 1 h. Sections were incubated with a rabbit polyclonal antibody against c-Fos (1:5,000, sc-253, Santa Cruz Biotechnology, Santa Cruz, CA, United States) diluted in PBS containing 1% bovine serum for 48 h at 4°C on an agitator. After rinsing in PBS, sections were incubated with a biotinylated goat anti-rabbit IgG (1:1,000, AP132B, Millipore, Temecula, CA, United States) then with horseradish peroxidase conjugated streptavidin (1:2,000, S2438, Sigma-Aldrich, Saint Louis, MO, United States). Both incubations were placed on an agitator at 4°C overnight. Following rinsing, the sections were immersed in 0.05 M Tris-HCl buffer, pH 7.6, containing 0.05% 3,3’diaminobenzidine (DAB), 0.01% H₂O₂, and 0.6% nickel ammonium sulfate for 2–5 min at room temperature. Finally, the sections were mounted on gelatin-coated glass slides, processed with counter-staining with neutral red, dried, dehydrated, and covered with a cover slip, using DPX, for light microscopy.

When subcutaneous tumors reached a diameter of approximately 8 mm, tumors were irradiated with 30 Gy of X-rays at a rate of 3.9 Gy/min with a TITAN-320 X-ray generator (Shimadzu, Kyoto, Japan). Other parts of the mouse body were covered with a brass shield. On post-exposure days 1, 3, and 7, tumors (n = 3 per time point) were sampled and fixed in 10% (v/v) neutral buffered formalin and embedded in paraffin for sectioning. Nontreated tumors were used as controls. Sections (thickness, 1 μm) were immunostained with antibody 3–6 (diluted 1:200) followed by horseradish peroxidase-conjugated anti-rat immunoglobulin from a kit (BD, Franklin Lakes, NJ, USA). Nuclei were counterstained with hematoxylin.
To compare antibodies 3–6 and 12–2–7, six subcutaneous A375 tumors were fixed with 4% paraformaldehyde overnight in 0.1 M sodium phosphate (pH 7.2). After dehydration in ethanol, the tissues were embedded in polyester wax. Adjacent sections were immunostained with anti-TNC antibody 3–6 (6 μg/mL) or 12–2–7 (8 μg/mL) as the primary antibody; the secondary antibody was goat anti-rat IgG biotin conjugate (SC-2041, Santa Cruz Biotechnology, Dallas, TX, USA; diluted 1:1000). A streptavidin-peroxidase polymer (S2438, Sigma; diluted 1:1000) served as the detection reagent. Coloring was done with diaminobenzidine, and nuclei were stained with methyl green.

Derivatized cell lysates were separated by 10% SDS-PAGE gels and transferred to nitrocellulose membranes, and probed using antibodies against p-Src Tyr 416 (1:400; 2101S), p-Src Tyr 527 (1:400; 2105S), Src (L4A1; 1:1000; 2110S), FLAG (DDK; 1:1000; 2368S) (Cell Signaling), or streptavidin peroxidase polymer ultrasensitive (1:10,000; S2438; Sigma). Primary antibodies were probed with rabbit- or mouse-specific secondary antibodies conjugated with HRP (Cell Signaling) and detected by enhanced chemiluminescence (Pierce). Membranes were imaged with Amersham Imager 600 (GE Healthcare) and band densities were quantified using ImageQuant TL (v8.1.0.0).