Rat anti mouse mac 2 antibody clone m3 38

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 (4μm thick) were cut using a Shandon Finesse 325 microtome. Resultant sections were stained with Giemsa, haemotoxylin and eosin or toluidine blue for subsequent analysis. For the PyMT study, sections from eight representative regions through each of the lungs were chosen and all tumours enumerated in the tissue. Giemsa stain was sufficient to discriminate the metastatic colonies from normal lung tissue. For immunohistochemistry, slides were deparaffinised, rehydrated through alcohols and stained using specific antibodies. To stain for CCL2 slides were boiled for 30 minutes in Tris-EDTA following blocking with 20% goat serum (Vector). Goat polyclonal anti-mouse CCL2 (M-18, Santa Cruz) was used as the primary antibody followed by an anti-goat HRP (Vector) secondary antibody. In the case of Mac2, antigen retrieval was not required. Slides were blocked using 20% goat serum (Vector) followed by rat anti-mouse Mac2 antibody (clone M3/38) (Cedar Lane), goat anti-rat IgG biotin (Vector) and finally Extravidin peroxidase LSAB reagent (Sigma).

Mouse kidney tissues were fixed in 4% neutral buffered formalin and embedded in paraffin. Periodic acid Schiff (PAS) or Masson's trichrome staining was performed on 3 µm kidney tissue sections. All glomeruli (between 50–110 glomeruli) in a single transverse section for each mouse were scored individually at 400× in a blinded manner by a pathologist and then averaged as previously described.^{50 (link)–53 (link)} Images were captured with an Olympus BX51 microscope and DP71 digital camera using cellSens Standard 1.12 image software (Olympus America, Center Valley, PA). Semiquantitative scores (0–4+) were assigned based on the masked readings as previously described.^{11 (link),47 ,54} Immunohistochemistry was performed using rat anti-mouse Mac-2 antibody (clone M3/38, Cedarlane, Burlington, NC), Wt-1 antibody (Cat #: SC-192, Santa Cruz Biotechnology, Santa Cruz, CA), cleaved caspase 3 antibody (Cat #: 9661, Cell Signaling Technology, Danvers, MA), on paraffin embedded sections. The number of glomerular macrophages, Wt-1 positive cells and apoptotic cells were counted in 15–40 glomeruli per section in blinded fashion under 40× magnification and averaged as described previously.^{11 (link),47 ,54}