Normal goat serum (ngs)

Colon sections (4 μm) cut from paraffin-embedded
intestine tissues were deparaffinized with xylene and rehydrated with ethanol.
For IHC, tissue sections were preincubated with 10% normal goat serum (ZSGB-BIO,
Beijing, China) in PBS (pH 7.5) and then incubated with primary antibodies
against BAHD1 (dilution: 1:200, Abcam, Cambridge, UK) overnight at
4 °C. Tissue sections were stained with HPR secondary
antibody (dilution: 1:1000, ZSGB-BIO, Beijing, China) for 1 h at
37 °C in an incubator. Immunoreactivity was detected
using a DAB kit (ZSGB-BIO, Beijing, China) and visualized as brown staining.

Five mice in each group were anesthetized with ether. They were perfused through heart with PBS containing 0.02% heparin, followed by 4% paraformaldehyde in PBS. Cerebral cortexes were quickly removed to a cold plate, then fixed overnight in 4% paraformaldehyde. Fixed samples were immersed in 30% sucrose for 3 days and embedded in OCT-compound. Serial frozen coronal sections (8 μm) were sliced at −20°C using a cryostat microtome. Thereafter, sections were permeabilized for 30 min in PBS containing 0.3% Triton X-100 and incubated for 30 min with normal goat serum (ZSGB-BIO, Beijing, China) to block nonspecific binding of antiserum.
For immunofluorescence staining, sections were incubated with rabbit antibodies against ZO-1 (Millipore, CA, USA) and occludin (Zymed, MA, USA), and mouse antibody against GFAP (Millipore, CA, USA) at 4°C overnight. On the following day, goat anti-rabbit FITC and goat anti-mouse TRITC conjugated secondary antibodies (ZSGB-BIO, Beijing, China), were added and incubated for 30 min at 37°C in a dark room. Finally, the frontoparietal region was observed under a fluorescence microscope (Olympus BX50). Images were captured using a digital camera system (Olympus SC35). The relative fluorescence intensities of ZO-1 and occludin were quantified using image J software (NIH, USA).

Samples were fixed in 4% formalin, paraffin-embedded, and sectioned (4 µm). After de-waxing and rehydration, the sections were incubated with 0.01 M citrate buffer for 20 min at 95 °C for antigen retrieval. Endogenous peroxidase activity and non-specific antigens were blocked with 3% hydrogen peroxide (ZSGB-Bio; Beijing, China) and 10% normal goat serum (ZSGB-Bio) respectively, followed by incubation with primary antibody at 4 °C overnight. Sections were rinsed with phosphate buffered saline (PBS), treated with goat anti-rabbit secondary antibody (ZSGB-Bio), visualized using 3, 3′-diaminobenzidine (DAB, ZSGB-Bio) as substrate, and counterstained with hematoxylin (Beyotime; Haimen, China). Normal mouse serum was used as the negative control. Staining of cancer cells was scored as follows: 0, no staining; 1, weak staining in <50% cells; 2, weak staining in ≥50% cells; 3, strong staining in <50% cells; and 4, strong staining in ≥50% cells. The following primary antibodies (Abcam, Cambridge, UK) were used at the dilutions indicated: CCDC109B (1:200), HIF1α (1:200), Ki-67 (1:500), MMP2 (1:100) and MMP9 (1:200).