Goat polyclonal anti mouse ccl2 m 18

Lungs and other tissues were fixed in 4% paraformaldehyde overnight before transfer to 70% ethanol. Tissues were embedded in paraffin wax using the Shandon Citadel 1000 (Thermo Shandon). Sections (4-μm thick) were cut using a Shandon Finesse 325 Microtome. Resultant sections were stained with Giemsa, H&E, or toluidine blue for subsequent analysis. For the PyMT study, sections from eight representative regions through each of the lungs were chosen, and all tumors were enumerated in the tissue. Giemsa stain was sufficient to discriminate the metastatic colonies from normal lung tissue. For immunohistochemistry, slides were deparaffinized, rehydrated through alcohols, and stained using specific Abs. To stain for CCL2, slides were boiled for 30 min in Tris-EDTA after blocking with 20% goat serum (Vector Laboratories). Goat polyclonal anti-mouse CCL2 (M-18; Santa Cruz Biotechnology) was used as the primary Ab, followed by an anti-goat HRP (Vector Laboratories) secondary Ab. In the case of Mac-2, Ag retrieval was not required. Slides were blocked using 20% goat serum (Vector Laboratories), followed by rat anti-mouse Mac-2 Ab (clone M3/38) (CEDARLANE), goat anti-rat IgG biotin (Vector Laboratories), and finally ExtrAvidin-Peroxidase labeled streptavidin-biotin reagent (Sigma-Aldrich).