Anti cytokeratin 5

Mammary tumors and lungs were collected and processed as previously described [32] (link), [33] . Immunohistochemistry was performed as previously described [32] (link). The number of lung metastases was determined in two independent histological sections from each mouse and then averaged. Primary antibodies were used at a dilution of 1∶200 and were obtained from the following sources, anti-Ki67, anti-cytokeratin 5 and anti-cytokeratin 14 (Abcam, Cambridge, MA), anti-cytokeratin 18 (Research Diagnostics Inc, Flanders, NJ), and anti-cytokeratin 8 (Fitzgerald Industries International Inc, Concord, MA). Primary antibodies were detected using a 1∶200 dilution of the appropriate secondary antibody and Sigma Fast 3,3′-diaminobenzidine tablets (Sigma, St. Louis, MO). Ki67 immunohistochemistry was quantified using Positive Pixel Count software v9 (Aperio, Vista, CA) following slide scanning on a ScanScope CS slide scanner (Aperio, Vista, CA).

For analysis of thymic medulla and cortex by immunohistology, thymi from GCV treated TK⁻ and TK⁺ mice were fixed in 4% formalin and embedded in paraffin blocks. Sections (5 μm) were stained with hematoxylin and eosin (H&E) and examined by light microscopy. For immunofluorescence, serial sections (5 μm) from OCT-embedded frozen tissues or primary cultured cells were fixed in cold acetone or 4% polyoxymethylene and blocked in PBS/1% BSA, washed in PBS/0.05% Tween and incubated with optimal dilutions of fluorochrome-conjugated antibodies: Alexa Fluor® 488 anti-I-A/I-E, anti-CD31-PE (Biolegend), anti-CD11c-PE, anti-CD11b-FTIC (BD Pharmingen), and anti-F4/80-PE (eBioscience), or with first Abs: anti-cytokeratin 5, anti-FSP1, anti-ER-TR7 (Abcam), anti-cytokeratin 8 (Tromal-1; Developmental Studies Hybridoma Bank), Biotinylated UEA-1, anti-vimentin (BD Pharmingen), anti-α-SMA, anti-Pan-CK (Sigma, Cat no. C5992), anti-CD140a/PDGFRα (R&D Systems) and anti-MTS15 Ab for 2 h at room temperature before washing and incubating with secondary reagents: Alexa Fluor® 546 Goat anti-Rabbit/mouse IgG (H+L), Alexa Fluor® 488 Goat anti-Rat IgG (H+L) (Invitrogen), Dylight 488 Goat anti-Rabbit/mouse IgG (ZSGB-Bio) and streptavidin-PE (BD PharMingen). Control slides were incubated with isotype-matched Ig. Images were acquired with a two-photon microscopy (Carl Zeiss, Inc.).