After paraffiniza tion, three to five micron thick sections were cut and mounted on slides. Imaging and analysis Digital images were taken of three fields per gland from three glands at 200 �� or 400 �� total magnification. For epithelial U0126 content determination, a grid of 360 boxes was overlaid on 200 �� images Inhibitors,Modulators,Libraries and boxes containing epithelial cells were counted. For IHC quantification, NIH ImageJ was used with a cell counter plug in to manually count positively stained mammary epithelial cells vs. total epithelial cells in multiple fields. Anno tated regions were drawn on each digital H E image using a pen tablet for area calculations by determining epithelial pixel count relative to the entire gland, and selecting regions of interest for digital IHC analysis.
For digital IHC quantification, slides were scanned at 40 �� magnifi cation using a whole slide scanner fitted with a 20x 0. 75 Plan Apo objective lens. Images were saved in SVS for mat compressed with JPG2000 at 70% quality and retrieved from a secure server using whole slide image management software. For automated quantification of molecules visualized Inhibitors,Modulators,Libraries by IHC, five annotated regions were drawn on each slide using a pen tablet screen on whole slide images viewed at high resolution using the Aperio systems annotation software. To detect individual cells in tissue sections, a nuclear cell quantification image analysis algorithm was trained on control slides by defining the color vectors for the hematoxylin nuclear counterstain and primary positive chromagen DAB, mini mum and maximum size for nuclei, and threshold ranges for intensity of nuclear staining.
The analysis algorithm was trained to detect nuclei in four intensity ranges for cells with no positive staining, weak positive staining, medium positive staining, and strong positive staining. Analyses were performed on each Inhibitors,Modulators,Libraries annotated region using defined settings and nuclear count results were collected from each slide. Data were represented as an H score, which accounts for staining intensity and percentage of positively stained cells. The H score 1 2 3. Each H score represents five fields each from Inhibitors,Modulators,Libraries three mice per time point. Mammary epithelial cell enrichment Mammary glands were harvested and weighed. Follow ing disruption with scalpels, tissue homogenates were incubated at 37 C Inhibitors,Modulators,Libraries in digestion buffer, 100 U ml hyaluronidase.
Digested mammary tissue was pel leted and washed with Hams F12 DMEM 1% serum three times at 1, 500 rpm, then twice at 800 rpm. Cell pellets were lysed as in with the addition of Roche PhosStop and Complete tablets. Immunohistochemistry high throughput screening Formalin fixed paraffin embedded sections of mammary glands were deparaffinized with xylenes, and rehydrated through graded alcohols. Rehydrated sections were equilibrated in PBS and microwaved in antigen retrieval buffer.