Alexa fluor 647 goat anti mouse igg h l

For histological analysis, ileal tissues of WAS rats were fixed in formalin for 24 hours and then paraffin embedded. Sections of 4 μm were cut. The sections were deparaffinized and hydrated, and then stained with hematoxylin and eosin (H&E) solution. Villus length and crypt depth were measured using image J software. Analyses were done on five well preserved crypts per slide and on 8 rats from each group.
For immunofluorescence analysis, ileal tissues were paraffin embedded, sectioned, dewaxed and antigen retrieval performed, while organoids were O.C.T. compound (SAKURA, USA) embedded, frozened, sectioned and antigen retrieval performed. Then, ileal tissues or organoid sections were blocked in 5% normal goat serum for 1 hour, and incubated with primary antibodies (anti-E-cadherin, 1:100, Cell Signaling, 3195; anti-PCNA, 1:50, Santa Cruz Biotechnology, sc-56; anti-lysozyme, 1:200, DAKO, clone EC32117) overnight at 4 °C, followed by 1 hour of incubation with secondary antibodies (Alexa Fluor 647 goat-anti-mouse IgG H + L, 1:200, Jackson Immuno Research, 715–605-150; Alexa Fluor 647 goat-anti-rabbit IgG H + L, 1:200, Jackson Immuno Research, 111–005-045) at room tempeture. Then, the samples were counterstained with Hoechst 33342 (1:10,000, Sigma-Aldrich) for 10 minutes at room tempeture. Fluorescence images were captured using fluorescence microscopy (DXM, Nikon).

For the detection of the intracellular distribution of FAP, MPRIP, and YAP, cells (1 × 10⁴ cells/well) were seeded into six-well glass-bottom plates, fixed in 4% paraformaldehyde for 15 min, and then permeabilized with 0.2% Triton X-100 (PBS) for 10 min. Nonspecific binding sites were blocked with 1% BSA in PBS for 1 h. Cells were treated with a primary antibody specific for FAP (sc-65398, Santa Cruz Biotechnology), MPRIP (14396, Cell Signaling Technology, Beverly, MA) or YAP(14074, Cell Signaling Technology, Beverly, MA) overnight at 4°C. Thereafter, the cells were incubated with Alexa Fluor647-goat anti-mouse IgG (H + L) and Alexa Fluor488-goat anti-rabbit IgG (H + L) (Jackson ImmunoResearch, 115-605-003, 111-545-003). Herein, 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime Institute of Biotechnology, Shanghai, China) was used to stain nuclei before capturing images. The images were acquired using a confocal microscope (Zeiss, Oberkochen, Germany). The red fluorescence indicated FAP expression, the green fluorescence indicated MPRIP or YAP expression, and the blue fluorescence indicated the nuclei.