Ph 6.0 citrate buffer

A de‐identified human colonic adenoma tissue microarray (TMA) derived from the TCPS patient cohort was obtained. The TMA was sectioned (5 μm) prior to deparaffinization, rehydration and antigen retrieval using pH 6.0 citrate buffer (DAKO) at 105°C for 20 min followed by 10 min at room temperature. The slide was incubated in 3% hydrogen peroxide for 10 min to reduce endogenous background signal and subsequently blocked in 3% BSA/10% donkey serum in PBS for 30 min. Multiplexed Immunofluorescence imaging was completed by sequential antibody staining and dye inactivation as described [6 (link)]. Briefly, imaging was performed on 124 TMA cores (1 mm diameter) using a Cytell Slide Imaging System (GE Healthcare) at 20× magnification. Images of each core were 5435 × 4473 pixels with a pixel resolution of 0.325 μm. Exposure times were optimized for each antibody stain. Antibody reagents are described in Table S1. Dye inactivation was accomplished with an alkaline peroxide solution, and background images were collected after each round of staining to ensure fluorophore inactivation. Following acquisition, images were processed as described [6 (link), 13 ]. Briefly, DAPI images for each round were registered to a common baseline, and autofluorescence in staining rounds was removed by subtracting the previous background image for each position. Images were then tiled for each TMA core.

In WT mice, intestinal tissues from the jejunal–ilial junction were surgically removed from euthanized mice and flush-cleaned using PBS. In APC^1638N/+ mice, intestinal tumors were counted as described previously (5 (link)) and tumor and tumor-adjacent normal tissues were collected for further analysis. Tissues were fixed in 10% buffered formalin, paraffin-embedded, and sectioned at 5-μm thickness for hematoxylin and eosin (H&E) staining and immunostaining. Sections were deparaffinized, sequentially rehydrated, and H&E-stained using established procedures. For 8-oxo-dG and BrdU staining, rehydrated sections were treated with 2 M HCl for 20 min at 37 °C followed by neutralization with 0.1 M borate buffer for 10 min at room temperature (RT) and incubated overnight with either anti–8-oxo-dG or anti-BrdU antibodies at 4 °C. For immunostaining of other proteins, rehydrated sections were used for antigen retrieval in pH 6.0 citrate buffer (Dako) for 20 min. All sections were permeabilized in 0.3% Triton X-100 and blocked with 5% BSA at RT followed by incubation in specific primary antibody at 4 °C. Detail about the immunostaining procedure is provided in SI Appendix, Section H.