Ab207996

Tumor tissues were fixed in 4% formaldehyde, dehydrated with gradient concentrations ethanol, embedded into parrafin (YA0011, Solarbio) and cut into sections with a thick of 5 μm. Sections were retrieved in 10 mM sodium citrate buffer (pH 6.0, P0083, Beyotime) for 15 min at 94°C. Following cooling to room temperature, sections were blocked with 1% bovine serum albumin (BSA, ST2249, Beyotime) for 30 min and then incubated with primary antibodies against RAD54B (1:200, ab238579, Abcam), Ki-67, c-myc (1:1000, ab32072, Abcam) and MMP-7 (1:500, ab216631, Abcam) respectively. Subsequently, sections were incubated with biotinylation-labeled secondary antibody (1:1000, ab207996, Abcam), re-stained with hematoxylin, and captured under a light microscope (Olympus). The relative level of RAD54B, Ki-67, cmyc and MMP-7 was determined as the ratio of the number of positive cells to the total number of cells.

Total proteins were extracted from Swan 71 cells, villous tissues, or mouse placental tissues with RIPA lysis buffer on ice for 30 min. Protein concentrations were determined using BCA Protein Quantification Kit (Vazyme, Nanjing, China). Western blotting analysis was performed as described previously [62 (link)]. Primary antibodies included anti-β-actin (ab8226, 1:5000), anti-β-Tubulin (ab108342, 1:10000), anti-GAPDH (ab8245,1: 5000), anti-Caspase-3 (ab184787,1:1000), anti-Bcl-2 (ab182858, 1:1000), and anti-Caspase-2 (ab179520, 1:2500), all of which were purchased from Abcam (Cambridge, UK). Secondary antibodies included goat anti-rabbit immunoglobulin G (IgG) (dilution 1:10,000; ab207995, Abcam) and goat anti-mouse IgG (dilution 1:10,000; ab207996, Abcam). The intensities of Western blotting bands were quantified using Image J software with β-actin, Tubulin, or GAPDH as loading control. In some cases, proteins with different molecule weights were analyzed in one gel and one membrane. Subsequently, this membrane was cut into different parts based on the locations of protein bands and then stained with their corresponding antibody. Loading controls with different molecular weights from the target proteins were selected to avoid overlap or proximity. For clarity, only one loading control was shown in figures.

To perform immunofluorescence staining on the colon tissue sections, the frozen sections embedded in OCT were fixed in 8% neutral buffered formalin for 30 min. After fixation, the sections were washed three times with distilled water (ddH2O) to remove residual formalin. For the staining process, primary antibodies specific to the target proteins of interest, such as anti-mouse Muc2 (ab76774, Abcam), CRAMP (sc-66843, Santa Cruz), or β-Defensin 2 (ab203077, Abcam), Ki67 (ab16667, Abcam), IL-1β (AF-401-NA, R&D) antibodies and anti E. coli antibody (ab25823, Abcam), were applied to the sections. Following the primary antibody incubation, secondary antibodies conjugated with biotin (ab6720, ab208000, ab207997, ab207996, Abcam and BAF109, R&D), were applied to the sections followed with streptavidin-FITC (405202, BioLegend) or PE (405204, BioLegend), allowing for the visualization of the target proteins. The 6-diamidino-2-phenylindole (DAPI) was used to stain the cell nuclei. The fluorescence intensity of all immunofluorescence staining was measured by ImageJ.