Mouse on mouse m o m blocking reagent

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).

Frozen tumor tissue sections were fixated in 2% paraformaldehyde and permeabilized in methanol. After blocking first using Mouse on Mouse (M.O.M.) Blocking Reagent (Vector Lab) and then in PBS containing 10% goat serum for 1 h each, slides were incubated overnight with antibodies: mouse anti-human CEACAM1 ectodomain (5F4, 1:200), rabbit anti-human -L cytoplasmic tail of CEACAM1 (229, 1:200) [54 (link)] or mouse anti-mouse CC1, a kind gift of K. Holmes (University of Colorado). The following day the slides were washed and secondary antibodies were applied for 1 h (goat anti-rabbit, Alexa Fluor 488 labeled and goat anti-mouse, Alexa Fluor 555, both 1:200, Invitrogen) were visualized using an Olympus IX81 Inverted microscope with Q Imaging Retiga 2000R cooled CCD camera with Image Pro Plus version 6.3 imaging software and 40X magnification. For nuclei staining, Hoechst 33342 (blue) was used at final concentration of 1 μg/ml.