Rabbit anti occludin

Western blot (WB) was performed according to a previous method (22 (link)). Briefly, after animals were anesthetized and decapitated, spinal cord tissues of the lesion site were removed and collected immediately on ice (8 (link)). Then, samples were homogenized and lysed in RIPA buffer containing protease and phosphatase inhibitors. After centrifuging at 12,000 rpm for 10 min at 4°C, protein concentrations were determined using a BCA Assay Kit (Beyotime). Equal amounts of protein lysate (20 μg) were separated by 10% SDS-PAGE electrophoresis, followed by transferring onto PVDF membranes. After blocking in 5% fresh-non-fat skim milk prepared in TBST for 2 h at room temperature, membranes were incubated with the appropriate primary antibodies at 4°C overnight. Then, membranes were incubated with corresponding HRP-conjugated secondary antibodies for 2 h at room temperature after washing with TBST. Finally, protein bands were visualized with chemiluminescent HRP Substrate (Thermo Fisher) under Western Lightning-ECL (Bio-Rad, USA). The following primary antibodies were used: mouse anti-Histone H3 (citrulline R2+ R8 +R17) (Abcam; 1:1000), rabbit anti-ZO-1 (Abcam, 1:5000), rabbit anti-occludin (Abcam, 1:5000), rabbit anti-transient receptor potential vanilloid type 4 (TRPV4) (Abcam; 1:1000), and mouse anti-GAPDH (Zen-bio, 1:5000).

The anterior and posterior brain regions were homogenized and equal concentrations of homogenate protein were loaded in 4–20% gradient gels, followed by electrophoresis and semi‐dry transfer. The membranes were blocked using Odyssey blocking buffer followed by overnight incubation in 1:2500 rabbit anti‐Claudin 1 abcam (Cambridge, MA), 1:1000 rabbit anti‐ZO‐1 Invitrogen (Grand Island, NY), 1:1000 rabbit anti‐Aquaporin 4 (abcam), or 1:250 rabbit anti‐Occludin (abcam) with either 1: 2000 mouse anti‐GAPDH (abcam) or 1:2000 mouse anti‐β‐actin antibodies. After three washes (5 minutes each), the membranes were incubated in donkey anti‐rabbit IRDye 700 and donkey anti‐mouse IRDye 800 for 45 minutes. The membranes were scanned using Odyssey scanner and the bands were analyzed using Image Studio Lite Version 3.1 Li‐Cor (Lincoln, NE). Relative expression was calculated as the ratio of fluorescence intensity of target protein to the intensity of GAPDH or βActin and then further normalized to the corresponding expression in the normal pregnant rats.

For immunofluorescence staining, cells or spinal cord sections were fixed in 4% PFA for 15 min and subsequently permeabilized in 0.2% Triton X-100/PBS for 20 min. The samples were treated with 5% bovine serum albumin (BSA) for 1 h at room temperature to block nonspecific binding. The samples were incubated overnight at 4 °C with primary antibody specific for rabbit anti-ZO1 (1:50, Invitrogen), rabbit anti-claudin-5 (1:100; Invitrogen), rabbit anti-occludin (1:50; Abcam), mouse anti-CD31 (1:200, Invitrogen), mouse anti-NeuN (1:500, Abcam), and rabbit anti-GRP78 (1:200, Proteintech). Samples were washed thrice with PBS and incubated with Alexa 594- or Alexa 488-conjugated secondary antibodies (1:200, Jackson ImmunoResearch, USA) for 1 h in the dark. Finally, nuclei were labeled with DAPI and images were obtained under similar exposure time and conditions.

For immunofluorescence staining, paraffin embedded sections (5 μm) with colon tissues were hydrated, treated for antigen retrieval with citrate buffer (pH 6), and the slides of cells were fixed in 4% formalin for 30 min and then washed with PBS for 3 times. Then they were blocked with 10% donkey serum for 30 min at 37 °C and incubated with the corresponding primary antibodies overnight at 4 °C. Antibodies used were: goat anti-ZO-1 (1:200, arigo) and rabbit anti-occludin (1:200, abcam). After washing with PBS for 3 times, slides were stained with secondary antibodies for 1 h at room temperature. Secondary antibodies used were Alexa Fluor 488 conjugated donkey anti-goat IgG and Alexa Fluor 594 conjugated donkey anti-rabbit IgG (1:200, Antgene Biotech Co., Ltd. Wuhan, China). For Ulex europaeus agglutinin-I (UEA-I) staining, sections were incubated with rhodamine UEA-I for 1 h at 37 °C. The nuclei were stained with DAPI (Beyotime Biotech, China) for 8 min at room temperature. Images were acquired on confocal microscope (Nikon, Japan).
Immunohistochemistry (IHC) of colon tissues was performed using a VECTASTAIN Elite ABC kit and a DAB Detection kit (Boster Biological Technology Co., Ltd) following the manufacturer’s instructions with an anti-F4/80 antibody (ARG55738, arigo).

Lysed cells and tissues were centrifuged at 13,000 x g for 30 min at 4°C after being incubated in cold RIPA buffer for 30 min. A BCA assay reagent (Beyotime) was used to calculate the protein levels of the lysates. Rabbit anti-occludin (1:1,000, Abcam), rabbit anti-ZO-1 (1:1,000, Abcam), and rabbit anti-β-actin (1:1,000, Abcam) were employed as the main antibodies. The chemiluminescent technique was used to identify proteins, and ImageJ was used to quantify them.

ARPE-19 cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Dulbecco’s Modified Eagle’s Medium: nutrient mixture F12 (hereafter named DMEM:F12), fetal bovine serum (FBS), bovine serum albumin (BSA), trypsin-EDTA, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium bicarbonate, gentamycin, phosphate-buffered saline (PBS), penicillin, streptomycin, 4′,6-diamidino-2-phenylindole (DAPI), propidium iodide (PI), Tween-20 and PAP pen were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fluorescein isothiocyanate (FITC)-labeled annexin V (to bind PS), annexin V-binding buffer containing 10 mM HEPES (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl₂, were purchased from Becton Dickinson Biosciences (BD), Belgium. Minimum essential medium (MEM) was purchased from Invitrogen (Carlsbad, CA, USA). Pipettes, 25 cm² flasks, 15 mL and 50 mL centrifugation tubes, 1 L glass bottles, and pipette tips were supplied by VWR International (West Chester, PA, USA). Vacuum filtration rapid filter mix was supplied by BioNordika (Oslo, Norway). Mouse anti-RPE65, rabbit anti-occludin, FITC-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG antibodies were obtained from Abcam (Cambridge, UK). Mouse anti-ZO-1 and Alexa Fluor 568 phalloidin were purchased from Life Technologies (Carlsbad, CA, USA).

Protein extraction and analysis were carried out according to the procedure of Hu et al. (29 (link)). The protein from jejunal mucosa was extracted by ProteoJET Total Protein Extraction Kit (Fermentas, Hanover, MD, USA) and its content was determined by BCA Protein Assay Kit (Beyotime, Shanghai, China). The protein was separated by 10% Sodium dodecyl-sulfate polyacrylamide electrophoresis (SDS-PAGE), and then transferred to an activated polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked by 5% BSA-TBST for 60 min, washed with TBST for 3 times, and then incubated overnight at 4°C using an appropriate primary antibody. The primary antibodies include Rabbit anti occludin (1:200, Abcam) and Rabbit anti-β-Actin (1:1000, Abcam). After incubation with a goat anti-rabbit IgG secondary antibody for 60 min, the target bands were displayed by Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) and analyzed by Quantity One Software (BioRad Laboratories, Hercules, CA, USA). The target protein-to-β-actin protein ratio was used to express the relative abundance of occludin in jejunal mucosa.