Anti zo 1 antibody

Sections from the distal small intestine from each treatment group were fixed in 10% formalin and embedded in paraffin wax. A minimum of 10 sections from each of the 5 animals per group were analyzed by immunofluorescence microscopy. Representative images are shown. For immunostaining with Claudin-1 antibody, 8 μm tissue sections were deparaffinized with xylene and tissue was rehydrated through a series of graded alcohol and then processed for antigen retrieval using citrate antigen retrieval buffer (DAKO). Tissue sections were stained with polyclonal rabbit antibody against Claudin-1 (Invitrogen) in PBS with 1% bovine serum albumin (BSA) overnight at 4°C. For immunostaining with anti-ZO-1 antibody, intestinal tissue was frozen in TFM tissue freezing media, and later 5 μm tissue sections were used. Sections were fixed with 1% paraformaldehyde for 10 min at room temperature and incubated with anti-ZO-1 antibody (Invitrogen). After washing, sections were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (Invitrogen); for F-actin staining, sections were incubated with Alexa 555 conjugated phalloidin (Invitrogen) for 1 h at room temperature. Sections were mounted under coverslip using ProLong Gold antifade reagent with DAPI (Invitrogen). Stained sections were imaged using a fluorescence microscope (Leica Microsystems, Germany).

hRPE were fixed in 4% PFA for 10 min at room temperature, followed by three 10 min washes with PBS for 5 min. Cells were then washed in PBS^−/− (Gibco) with 0.3% Triton™ X-100 (wash buffer) for five minutes, thrice, before blocking in 1% BSA, 3% FBS, 0.3% Triton X-100 in PBS^−/− at room temperature for 20 min. Blocking buffer was then replaced with anti-ZO1 antibody (1:100, Invitrogen, cat. 40–2200) in blocking buffer in 4˚C overnight. On the following day, wash buffer was replaced three times every five minutes before the appropriate secondary antibody (anti-rabbit IgG H&L Alexa Fluor®647, 1:400, Invitrogen, cat. A-31573) blocking buffer was applied for 1 h at room temperature. After three more washes, slides were mounted using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories). Confocal images were acquired with Carl Zeiss LSM 780.

Lysates from the corpus callosum region were prepared in Pro-PREPTM Protein Extraction Solution (Boca Scientific). Samples with equal volumes of SDS sample buffer (Novex) and 2-ME were heated at 95°C for 5 min, then each sample (20 µg per lane) was loaded onto 4–20% Tris–glycine gels. After electrophoresis and transferring to polyvinylidene difluoride membranes (Novex), the membranes were blocked in Brockace (AbD Serotec) for 60 min at room temperature. Membranes were then incubated overnight at 4°C with anti-ZO-1 antibody (1∶1000, Invitrogen), anti-occludin antibody (1∶1000, Abcam), anti-claudin5 (1∶1000, Abcam), anti-ERK1/2 (1∶1000, Cell Signaling), anti-phospho-ERK1/2 (1∶000, Cell Signaling) or anti-β-actin antibody (1∶5000, Sigma Aldrich) followed by incubation with peroxidase-conjugated secondary antibodies and visualization by enhanced chemiluminescence (Amersham).

Immunocytochemistry was performed by modifying procedures that have been previously described [23 (link)]. The samples were fixed with methanol at 25 °C and then soaked in 3% BSA blocking solution, also at 25 °C. The samples were incubated with anti-ZO-1 antibody (1:400, Invitrogen), anti-claudin-5 antibody (1:400, Invitrogen), or anti-FoxO3a antibody (1:400, Cell Signaling Technology, Inc., Danvers, MA, USA) overnight and then with secondary antibody labeled with Alexa 488 (1:500, Invitrogen). Finally, the samples were incubated with Hoechst 33258. All samples were observed under a Nikon C2 confocal microscope and all cells were pretreated with 30 μg/mL HSP 30 min before hypoxia exposure.

The immunohistochemistry of ZO-1 tight junction protein double labelled with contractile protein α-SMA was performed on the same bladder tissues. The slides were subjected to antigen retrieval, as described above. Then they were then incubated with 10% goat serum for 30 min to block unspecific binding sites of secondary antibody followed by incubation with anti-ZO-1 antibody (Invitrogen 61–7,300, 1:100) and anti-α-SMA antibody (Dako M085129-2, 1:200) overnight at room temperature. Following the incubation with the primary antibody, the slides were washed (3 × 10 min) in TBS and tagged with a secondary fluorescent antibody for 2 h at room temperature (ZO-1; Alexa Fluor 594, 1:200, ab150080, α-SMA; Alexa Fluor 488, 1:200, ab150117). After the secondary antibody, the slides were washed again with TBS (3 × 10 min) and then mounted with DAPI.
The immunoreactive images were captured using the Neurolucida microscope, 40× objectives, and analyzed using ImageJ. ZO-1 scoring was conducted using the criteria in Table 2 at 40× magnification (n = 5 for each group). The mean score was determined for each treatment group and the results were expressed as mean ± SEM. The level of α-SMA immunoreactivity in the suburothelium was estimated as strong (+++), moderate (++), and weak (+).