Quantification of LGR5+ Cells in Jejunum

To quantify LGR5+ cells in jejunal tissues, formalin-fixed tissue sections were stained with anti- LGR5 antibodies using the Mach3 Rabbit AP-Polymer Detection Kit (Biocare Medical, USA) as performed earlier (31 (link)). The paraffin-embedded tissue sections were deparaffinized and epitope retrieved by heating the tissue sections in low pH citrate buffer (Vector Laboratories, USA) using a microwave. After blocking with a serum-free protein blocker (Agilent Dako, USA) for 30 min, the tissue sections were incubated with anti-LGR5 primary antibodies (Supplementary Table 1). The negative control slide consisted of rabbit Ig fractions (R&D Systems, USA) used at the same isotype and concentration as LGR5 antibodies to determine the background intensity and staining. The tissue sections were subsequently treated with the kit’s probe and polymer and finally developed using BCIP/NBT (Agilent Dako) chromogen system. The slides were then mounted with Vecta Mount AQ (Vector Laboratories). An average of 19–20 view fields (0.116mm²/view field under a 400 x magnification) were enumerated in each of the slides to quantify LGR5+ cells manually. The tissue sites for this evaluation were selected randomly. Tissues were analyzed by two different blinded individuals to avoid bias and averaged prior to the statistical analysis.