Serum free protein blocking agent

Slides were baked and deparaffinized in xylene and alcohol series followed by heat-mediated antigen retrieval using sodium citrate buffer. Tissue sections were then blocked with the Mouse On Mouse (M.O.M.) blocking reagent for 1 h (Vector Labs) and serum-free protein blocking agent (Dako) and then incubated with mouse mAb for Phospho-Histone H2A.X (Ser139, 9F3, Abcam) with rabbit anti-Lysozyme (EC 3.2.1.17, DAKO) for jejunum sections or rat anti-TROMA1 (DSHB) for ovarian tumor sections at 4 °C overnight. Slides were washed in 1X PBS and incubated with Alexafluor 594 conjugated anti-mouse secondary antibody, Alexaflour 488 anti-rabbit or Alexaflour 488 anti-rat for 30 min at 37 °C. 1X PBS containing 0.1% BSA and 0.2% Triton X-100 were used to dilute the antibodies. The slides were then DAPI stained. Images were captured as Z-stacks using a fluorescence microscope (Leica DMi8). Thirty one slices with a step size of 0.4 μm (z-direction) between slices were captured/z-stack to map the entire nucleus^{40 (link)}. At least three 60× images were collected randomly for each time point. Maximum intensity projections of the captured Z-stacks were analyzed for γ-H2AX foci. An average of 10 CBC cells, located in between lysozyme positive Paneth cells per mouse were analyzed for the number of punctate γ-H2AX foci in jejunal circumferences using the Fiji ImageJ software (n = 4 mice/ group).

Paraffin‐embedded tissue sections were deparaffinized through a xylene/graded ethanol series following standard histochemical procedures; antigen retrieval was performed by heat treating in a citrate buffer (Dako North America, Inc., Carpinteria, CA) for 20–30 min using a pressure cooker. Slides were blocked with a peroxidase‐blocking solution (Dako), preincubated with a serum‐free protein blocking agent (Dako), and incubated with an anti‐Ki67 antibody (Abcam, Cambridge, MA) diluted in an antibody diluent reagent (Dako) at 4°C overnight. Primary antibody binding was revealed using an Envision+ HRP system followed by 3,3′‐diaminobenzidine tetrahydrochloride (Dako) following the manufacturer's recommendations, and the slides were counterstained with hematoxylin prior to mounting. For quantification of Ki67 staining, slides from four representative animals per group were digitized using a Chromavision ACIS II scanner, and the total and Ki67‐positive nuclei from cyst‐lining epithelial cells from 40 to 50 cysts per animal were counted manually.