Ab307799

WB was exploited to detect the protein expression of ZO‐1, Occludin, IL‐17A, and IL‐17F in NCM460 cells. Proteins were extracted for SDS‐PAGE protein electrophoresis. Then the proteins were transferred to PVDF membrane at 4°C for 2 h. Samples were blocked with 5% skim milk TBST and shaken at room temperature for 2 h. The internal reference was GAPDH. Then antibodies (Abcam) were added, incubated at 4°C overnight with shaking, including ZO‐1 antibody (ab307799, 1/1000), Occludin antibody (ab216327, 1/1000), IL‐17A antibody (ab79056, 1/1000), and IL‐17F antibody (ab187059, 1/1000). Then, the membrane was washed and incubated with anti‐IgG secondary antibodies conjugated with horseradish peroxidase. Finally, ECL solution was added for developing, and images were captured with Tanon 5200 Chemiluminescent Imaging System (Tanon).

For hematoxylin and eosin (H&E) staining, coronal sections of brain tissues, 20 µm thick, were stained using H&E, with incubation of 2 min for hematoxylin and 1 min for eosin. Sections underwent dehydration and permeabilization before microscopic observation (BX63, Olympus, Japan) (Li et al. 2020b (link)).
For immunofluorescence assay, fixed cells or tissue sections were treated to allow penetration of primary antibodies targeting ZO-1 (ab307799, Abcam, UK), Occludin (ab216327, Abcam, UK), Claudin-5 (ab131259, Abcam, UK), and CD31 (Sc-376764, SANTA CRUZ, US), followed by incubation with Alexa Fluor-conjugated secondary antibodies and DAPI staining. Confocal microscopy facilitated the visualization of these markers (Li et al. 2019 (link)).
The immunohistochemistry protocol entailed the use of antibodies specific to NeuN (ab177487, Abcam), TNF-α (ab307164, Abcam), and IL-1β (ab283818, Abcam). Following secondary antibody application and SABC amplification, the DAB chromogen revealed the localization of target proteins, which was counterstained with hematoxylin. Observations were made under an upright microscope (BX63, Olympus, Japan) (Li et al. 2020b (link)).

The expression of ZO-1 was detected using immunofluorescence staining as described before [23 (link)]. The mouse brain tissue was fixed with 4% paraformaldehyde and dehydrated with 30% sucrose, followed by being embedded and sliced into sections (20 μm). The sections were incubated with blocking solution (5% horse serum and 0.1% Triton X-100) for 90 min, and then incubated with ZO-1 primary antibody (1:100, Cat#ab307799, Abcam, USA) at 4° C overnight. After adding the corresponding fluorescent secondary antibody (1:200, Abcam, USA), the sections were incubated for 90 min. The slides were sealed with nail polish and air-dried in a Fume Hood before being photographed using a fluorescence microscope (Leica, Wetzlar, Germany).