Mouse anti claudin 5

Cells were lysed with RIPA buffer (50-mM Tris pH 7.4, 1% NP-40, 0.5% Na-deoxycholate, 1% SDS, 150-mM NaCl, 2-mM EDTA, 1 × protease inhibitor cocktail, and 1 × phosphatase inhibitor cocktail). Total protein levels were determined using the Bio-Rad protein assay kit, and equal amounts of proteins were loaded and separated on SDS-PAGE. After transferring to PVDF membrane (Millipore), proteins were detected using a standard immune-blotting technique. The following primary antibodies were used: mouse anti-claudin-5 (1:500, Invitrogen, USA, 35-2500), rabbit anti-ZO-1 (1:500, Thermofisher, USA, 61-7300), rabbit anti-caveolin-1 (1:1000, cell signaling, 3238S), and mouse anti-β-actin (Sigma, A5441, 1:2000). Target proteins were visualized using SuperSignal West Pico Plus Chemiluminescent Substrate (Thermo scientific). The density of target protein bands was quantified using NIH ImageJ software. The expression of target proteins was normalized to that of β-actin.

The antibodies used in this study were obtained from the following sources: Rabbit anti-ZO-1 (#8193), rabbit anti-claudin-3 (#341700), rabbit anti-VE-cadherin (#D87F2), rabbit anti-VCAM-1 (#12367), mouse PECAM-1 (#89C2) from Cell Signaling Technology (Danvers, MA, USA); mouse anti-E-selectin (#S 9555), β-actin (#A5441) from Sigma-Aldrich (St. Louis, MO, USA); donkey anti-rabbit (#NA934) and sheep anti-mouse (#NA931) HRP-linked secondary antibodies from GE Healthcare (Piscataway, NJ, USA); mouse anti-claudin 5 (#35-2500), goat anti-rabbit (#A11008) and anti-mouse (#A21422) conjugated to Alexa Fluor® 488 and 555 from Invitrogen (Camarillo, CA, USA). Sterile cultureware was obtained from Fisher Scientific (Pittsburgh, PA, USA), while other reagents and chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Bio-Rad laboratories (Hercules, CA, USA).

The antibodies used in this study were obtained from the following sources: Rabbit anti-ZO-1 (#D7D12) and anti-VE-cadherin (#D87F2) from Cell Signaling Technology (Danvers, MA, USA); Rabbit anti-GLUT-1 (#ab15309) and anti-SGLT-1 (#ab14686) from Abcam (Cambridge, MA, USA); Rabbit anti-GLUT-4 (#sc-7938) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Donkey anti-rabbit (#NA934) and sheep anti-mouse (#NA931) HRP-linked antibodies from GE Healthcare (Piscataway, NJ, USA); Mouse anti-Claudin 5 (#35-2500), goat anti-mouse (#A11001) and anti-rabbit (#A21428) conjugated to Alexa Fluor® 488 and 555 from Invitrogen (Camarillo, CA, USA). Sterile culture ware was obtained from Fisher Scientific (Pittsburgh, PA, USA), while other reagents and chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Bio-Rad laboratories (Hercules, CA, USA). Fluorescein isothiocyanate (FITC) and Rhodamine B isothiocyanate (RITC) dextrans were purchased from Sigma-Aldrich, while Cascade Blue®-dextran was obtained from Invitrogen (Eugene, OR, USA).

The animals under anesthesia were infused via the left ventricle with 4% paraformaldehyde in 0.01 M PBS (pH 7.4). Colon segment 5 cm proximal to anus was removed and post-fixed with the same fixative for 12 hours, and cryoprotected in 30% sucrose in PBS for at least 24 hours at 4 °C. The colon with mucosal injure was sliced in 10 μm thick using a cryostat (CM1800; Leica, Bensheim, Germany). To evaluate the cell junction in colonic mucosa, immunofluorescence staining of occludin and claudin-5 was performed. For this purpose, slices were incubated with the following primary antibodies overnight at 4 °C: mouse anti-claudin-5 (1: 100, Invitrogen, Camarillo, CA, USA), mouse anti-occludin (1: 50, Invitrogen, Camarillo, CA, USA). After washing, sections were incubated with dylight 549-labeled goat anti-mouse IgG (KPL, Gaithersburg, MD, USA) for 2 hours at room temperature. Hoechst 33342 (BD Biosciences Pharmingen, San Jose, CA, USA) was applied to stain nucleus. F-actin in colonic tissues was stained with phalloidin (1: 40, Abcam, Cambridge, UK). All sections were photographed under a laser scanning confocal microscope (TCS SP5, Leica, Mannheim, Germany).

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.

For evaluation of the expression of tight junction (TJ) proteins claudin-5 and ZO-1 in cerebral microvessels, immunofluorescence staining was performed. The tissue preparation and staining was performed as per described protocol (Li et al., 2018 (link)), and the primary antibodies applied included: mouse anti-claudin-5 and anti-ZO-1 (1:50, Invitrogen, Camarillo, CA, United States), and rabbit anti-vWF (1:100, Millipore, Temecula, CA, United States). The brain sections were mounted, coverslipped, and photographed under a laser scanning confocal microscope (TCS SP5, Leica, Mannheim, Germany), as described previously (Li et al., 2018 (link)).