Anti olfm4 antibody

Cells were lysed in radioimmunoprecipitation assay lysis buffer (Beyotime) supplemented with protease inhibitor (cOmplete Tablets, Mini, EDTA-free, Roche, Basel Switzerland). Proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes. Primary antibodies were used for immunoblotting: anti-OLFM4 antibody (Cat No. 14369D, Cell Signaling Technology, Danvers, Massachusetts, USA), caspase-3/p17/p19 (Cat No. 66,470–2-Ig, Proteintech, Rosemont, IL, USA), cleaved caspase-3 (Cat No. 9664, Cell Signaling Technology), caspase-9/p35/p10 (Cat No. 66,169–1-Ig, Proteintech), Bax (Cat No. 60,267–1-Ig, Proteintech), Bad (Cat No. 10,435–1-AP, Proteintech), and ARL6IP1 (Cat No. ab24228, Abcam, Cambridge, UK). β-actin (Cat No. 66,009–1-Ig, Proteintech) was used as a loading control. The membranes were blocked with 5% skim milk diluted in TBST and incubated with primary and then secondary antibodies. Finally, the blots were incubated in enhanced chemiluminescent reagent (Affinity Biosciences, Cincinnati, OH, USA) and visualized with the ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA).

For tissue immunofluorescence staining, paraffin sections of the ileum were hydrated, treated with citrate buffer (pH 6.0) for antigen retrieval, permeabilized with 0.3% Triton, blocked with 10% donkey serum, and incubated with the following primary antibodies and corresponding secondary antibodies (all 1:200 dilution): anti-Lysozyme antibody (Abcam, ab108508), anti-Muc2 antibody (Genetex, GTX100664), anti-chromogranin A antibody (Abcam, ab45179), anti-Ki67 antibody (Servicebio, GB111141), anti-GFP antibody (Abcam, ab290), anti-β-catenin antibody (Abclonal, A19657), UEA-I Rhodamine (Vector, RL-1062-2), Alexa Fluor 488 conjugated donkey anti-rabbit IgG (Antgene, ANT024S), Alexa Fluor 488 conjugated donkey anti-goat IgG (Antgene, ANT025S), and Alexa Fluor 594 conjugated donkey anti-rabbit IgG (Antgene, ANT030S). Nuclei were stained with DAPI.
For organoid immunofluorescence staining, the culture medium was removed and the wells were washed with PBS. Organoids were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton, blocked with 10% donkey serum, and incubated with the corresponding primary and secondary antibodies.
IHC staining of the ileum tissue was performed using a SABC-AP IHC kit for rabbit IgG (Boster Biological Technology Co., Ltd.) according to the manufacturer’s instructions. The primary antibody used was the anti-Olfm4 antibody (Cell Signaling, 39141).

Total proteins from tissues and organoids were harvested using RIPA Lysis Buffer (Beyotime) with phenylmethyl sulfonyl fluoride (PMSF) protease inhibitor and phosphatase inhibitor. Samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes, followed by blocking with 5% skimmed milk for 1 h. The membranes were then incubated with primary antibodies and the corresponding secondary antibodies (all 1:1000 dilutions): anti-Lgr5 antibody (Abcam, ab75850), anti-Olfm4 antibody (Cell Signaling, 39141), anti-GAPDH antibody (Abclonal, AC001), anti-GPR41 antibody (Abclonal, A12636), anti-GPR43 antibody (Proteintech, 19952-1-AP), anti-β-tubulin (Abclonal, AC015), anti-Axin2 antibody (Proteintech, 20540-1-AP), anti-β-catenin (Abclonal, A19657), anti-Wnt3 antibody (Abclonal, A9328), and HRP conjugated Goat anti-rabbit IgG (Antgene, 72-8067). The bands were detected using a chemiluminescence imaging system (UVP) with ECL chemiluminescence detection kit (Vazyme). The relative fold change in protein expression was normalized to that of GAPDH or β-tubulin.