Rabbit anti occludin

The MEC monolayers were washed three times with PBS 1× (Life Technologies) and gently dissociated from the insert filters followed by a 20-min fixation in paraformaldehyde (PFA) 4 % (w/v) (Sigma Aldrich, Saint-Louis, MO, USA) prior to immunocytochemistry. After three washes with PBS 1× (Life Technologies), the cells were pre-incubated for 30 min at room temperature (RT) with blocking buffer containing BSA 3 % (PAA Laboratories, Velizy-Villacoublay, France) in PBS 1×. The MEC monolayers were stained for 1 h in PBS 1× containing BSA 1 % (PAA Laboratories), with saponine 0.1 % (Sigma Aldrich) for membrane permeabilization, with a rabbit anti-occludin 1.5 μg/mL (Life Technologies). Cell nuclei were labeled with Hoechst 33342 1/1000 (Life Technologies) in co-incubation with a donkey anti-rabbit Alexa Fluor 488 secondary antibody (Jackson Immunoresearch, West Grove, PA, USA). Cells were washed and mounted in Prolong Gold antifade mounting medium (Life Technologies). The mounted slides were observed with a Leica TCS SP2 confocal microscope (Leica Microsystems, Heidelberg, Germany). High-magnification images were acquired using a 63X HCX PL APO oil immersion objective and analyzed using the NIH ImageJ software (version 1.49o for Mac).

The heart sections were incubated in blocking solution for 1 h at room temperature and then incubated at 4 °C overnight with one of the following antibodies: rabbit anti-VE-cadherin (1:200; Invitrogen Cat. #36-1900), goat anti-CD31 (1:200; R&D Cat. #AF3628), rabbit anti-zonula occludens-1 (1:200; Invitrogen Cat. #61-7300), rabbit anti-occludin (1:200; Invitrogen Cat. #71-1500), mouse anti-NG2 (1:100; Millipore Cat. #AB5320), rabbit anti-VCAM-1 (1:200; Santa Cruz Cat. #sc-13160), rabbit anti-MPO (1:50; Abcam Cat. #ab9535), mouse anti-CD68 (1:100; Abcam Cat. #ab955), rabbit anti-iNOS (1:200; Abcam Cat. #ab15323), rabbit anti-CD206 (1:100; Abcam Cat. #ab64693), or mouse anti-cTnT (1:200; Abcam Cat. #ab8295). Confocal images were captured at room temperature with ZEN software on an upright confocal microscope (LSM 700; Carl Zeiss) using the predefined ZEN software configurations for Alexa Fluor 546, Alexa Fluor 488, and DAPI.

After EA, the cerebral cortex of each group of rats were taken, total protein of samples was extracted, protein content was determined by BCA method, protein pre-denaturation, electrophoresis, gel cutting and membrane transfer were performed. 0.5% defatted milk powder was used as the closure solution, rabbit anti-β-actin (1:2500 Cell signal, 5125S) and rabbit anti-occludin (1:1000, Invitrogen, 71-1500), rabbit anti-NR1 (1:600 Abcam, ab17345) overnight at 4°C and rewarmed at 37°C, The membrane was incubated with an HRP-conjugated goat anti-rabbit/mouse (H + L) secondary antibody (1:5000 Bioker Biotechnology) diluted 1:5000 in TBST for 2 h at room temperature. Wash the membranes and add the chromogenic agent to use. Finally, protein bands were observed using the ImageQuant LAS 4000 system and semi-quantified using Image-J software.

For analysis of the tight junction composition, membrane protein fractions were prepared [23 (link)], and the expression of tight junction proteins discussed as being involved in macromolecule pathway regulation was quantified by immunoblotting employing primary antibodies against tricellulin (rabbit anti-Tric, Invitrogen, Dreireich, Germany, Cat.-No. 700191), occludin (rabbit anti-occludin, Invitrogen, Cat.-No. 711500), angulin-1 (rabbit anti-LSR, Atlas Antibodies, Bromma, Sweden, Cat.-No. HPA007270), and β-Actin (mouse anti-b-Actin, Sigma Aldrich, Cat.-No. A5441) as described [28 (link)]. Specific signals were quantified by chemiluminescence detection after incubation of the washed membranes with Lumi-Light plus (Hoffman La Roche, Basel, Switzerland) using luminescence imaging (Fusion FX7, Vilber Lourmat, Eberhardzell, Germany) and quantification software (Multi-Gauge V2.3, FujiFilm, Tokyo, Japan).

Briefly, 20 μg of cerebral cortex samples protein from each group was separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane, which was blocked with 5% non-fat milk. The membrane was incubated with the following primary antibodies in Tris–buffered saline Tween (TBST), pH 7.4, at 4°C overnight: HRP-conjugated rabbit beta-actin mAb (1:1000, Cell Signaling, 5125S), mouse anti-c-fos (1:2000, Invitrogen, 4700), rabbit anti-ZO-1 (1:1000, Invitrogen, 61–7300), rabbit anti-Occludin (1:1000, Invitrogen, 71–1500), mouse anti-Claudin 5 (1:1000, Invitrogen, 35–2500), mouse anti-GFAP (1:2000, Invitrogen, MA5-12023), rabbit anti-Iba1 (1:1000, Huabio, ET1705-78), and rabbit anti-Aquaporin (1:1000, Huabio, ER1903-87). The membrane was incubated with an HRP-conjugated goat anti-rabbit/mouse (H+L) secondary antibody (Bioker Biotechnology) diluted 1:5000 in TBST for 2 h at room temperature. The protein bands were viewed with the ImageQuant LAS 4000 system, and densitometry was quantified using ImageJ software.

HBMECs were fixed in 4% formaldehyde for 20 min at room temperature and permeabilized in 0.1% Triton X-100 in PBS for 15 min at 4 °C. Cells were incubated with antibodies such as rabbit anti-occludin (1:100, Invitrogen), mouse anti-claudin-5 (1:50, Invitrogen), or rabbit anti-zonula occludens-1 (ZO-1, 1:200, Invitrogen) overnight at 4 °C. The cells were incubated with secondary antibodies conjugated with Alexa Fluor for 1 h at room temperature. Actin filaments were monitored with rhodamine phalloidin (Molecular Probes) for 30 min. Cells were mounted using Dako mounting reagent and were observed using a fluorescence microscope (Zeiss; ×400).