mouse monoclonal primary antibodies against tight junction marker zo-1  (Thermo Fisher)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Oral administration of S -nitroso- l -glutathione (GSNO) provides anti-inflammatory and cytoprotective effects during ocular bacterial infections

doi: 10.1007/s00018-023-04963-w

Figure Lengend Snippet: GSNO treatment prevents the disruption of outer blood-retinal barrier. A–D Human retinal RPE cells (ARPE-19 cell line) were treated with GSNO (200 μg/mL) or PBS (control, C) for 1 h, followed by infection with S. aureus (SA) at MOI of 10 for 6 h. A The cells were fixed and immunostained with ZO-1 antibody (green) and nuclear stain DAPI (blue) and visualized under fluorescence microscope. Scale bar; 50 μm. B In another experiment cells were subjected to immunoblotting to quantify ZO-2 expression and qualification by densitometry analysis (C). The barrier properties were assessed by the FITC-dextran trans-epithelial permeability assay (D). The integrity of outer BRB was assessed using ex-vivo model and staining of RPE layer in choroid/RPE flat mount. E Eye cups from B6 mice were incubated in DMEM, followed by GSNO (200 μg/mL) treatment for 1 h and SA infection for additional 4 h. Following PFA fixation, eye cups were stained with ZO-1 and flat mounted to visualize under confocal microscope (blue, DAPI nuclear stain; red, ZO-1), Scale bar; 100 μm. The retinal lysates from oral GSNO treated (72 h time point) mice were subjected to immunoblotting to check ZO-2 expression (F) and quantitation by densitometric analysis using ImagJ (G) Densitometric analysis was done data are represented as mean ± SD. Statistical analysis was performed using ANOVA (*) p < 0.05 (**) p < 0.01 (***) p < 0.001 (****) p < 0.0001, ns; non-significant. Significance was compared between control, C vs SA samples and SA vs GSNO-treated samples. The results are represented from at least two independent experiments

Article Snippet: Following permeabilization and blocking, they were stained with rabbit polyclonal antibodies against mouse ZO-1 (1:200, Invitrogen) and visualized with Alexa 594 before flat mounting with Vectashield antifade medium on lysine-coated microscope slides.

Techniques: Disruption, Infection, Staining, Fluorescence, Microscopy, Western Blot, Expressing, Permeability, Ex Vivo, Incubation, Quantitation Assay

Journal: PLoS ONE

Article Title: Localization and Functional Characterization of the Rat Oatp4c1 Transporter in an In Vitro Cell System and Rat Tissues

doi: 10.1371/journal.pone.0039641

Figure Lengend Snippet: (A) Western blot analysis for Oatp4c1 expression. Protein lysates (50 µg per lane) from MDCKII cells transfected with pcDNA (empty vector) and Oatp4c1 were prepared from cells grown in monolayers. (B) Immunohistochemical analysis for Oatp4c1 expression. Paraformaldehyde-fixed paraffin-embedded cell sections were stained with PA1343. Color development with NovaRed signifies Oatp4c1 staining. All sections were counterstained with hemotoxylin. Rabbit IgG was used as a negative control. (C) Immunolocalization of Oatp4c1 in polarized MDCKII cells. Cells were double stained with Oatp4c1 (red) and ZO-1 (green). Nuclei were stained with DAPI (blue). Center image in the Oatp4c1 panel is a single optical section of the x–y plane while top and right images represent x–z and y–z planes, respectively, reconstructed from image stacks. The apical and basal sides can be demarcated by ZO-1 and the nuclei, respectively, in both x–z and y–z sections. (D) Western blot analysis of Oatp4c1 in proteins isolated from either the apical (AP) or basolateral (BL) membranes of polarized MDCKII cells. Surface proteins were isolated by biotin-streptavidin labeling on either the apical AP or BL compartment. Na + /K + -ATPase served as a BL marker to demonstrate the relative efficiency of AP and BL membrane separation. (Magnification (40×)).

Article Snippet: Mouse monoclonal antibody against ZO-1 (zo1-1A12) was from Invitrogen.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Immunohistochemical staining, Staining, Negative Control, Isolation, Labeling, Marker

Journal: Respiratory Research

Article Title: Nitric oxide gas phase release in human small airway epithelial cells

doi: 10.1186/1465-9921-10-3

Figure Lengend Snippet: Phenotypic markers of human small airway epithelial cells (SAECs) cultured at an air-liquid interface . Day 0 represents the first day of air-liquid interface which is generally 48 hours after seeding on the membrane, and all representative images were taken at day 7. A: Immunofluorescence imaging of MUC5AC (green) demonstrating the presence of mucous granules. B: Immunofluorescence imaging of β-tubulin IV (green) demonstrates the presence of ciliated cells. C: Immunofluorescence imaging of zonula occludens-1 (ZO-1) (green), a key protein present in the intercellular tight junctions. Cell nuclei were counterstained blue by DAPI. D: Transepithelial electrical resistance (TER) was measured every other day from 2 days of ALI. TER data represent the mean response from 2 different donors (12 monolayers from each donor). Scale bar: 10 μm.

Article Snippet: Mouse monoclonal antibodies against ZO-1 (1:250 dilution; Zymed Laboratories, South San Francisco, CA), β-tubulin IV (1:1000 dilution; Sigma, St. Louis, MO), and mucin 5AC (MUC5AC, 1:1000 dilution; Neo Markers, Fremont, CA) were diluted in PBS with 5% goat serum and incubated at 4°C overnight.

Techniques: Cell Culture, Immunofluorescence, Imaging

Journal: Molecular Vision

Article Title: Elevated pressure downregulates ZO-1 expression and disrupts cytoskeleton and focal adhesion in human trabecular meshwork cells

doi:

Figure Lengend Snippet: Western blot analysis of expression of ZO-1 in iHTM and GTM 3 at 12 h and 24 h of culture in a conventional incubator and in a 60 mmHg pressure system. Elevated pressure caused the expression of ZO-1 decreased in both iHTM andGTM 3 (*p<0.05).

Article Snippet: After blocking the cells with 5% bovine serum albumin (BSA), they were incubated overnight at 4 °C with mouse antibody against ZO-1 (1:50; Invitrogen, Carlsbad, CA) diluted in 1% BSA in PBS.

Techniques: Western Blot, Expressing

Journal: Molecular Vision

Article Title: Elevated pressure downregulates ZO-1 expression and disrupts cytoskeleton and focal adhesion in human trabecular meshwork cells

doi:

Figure Lengend Snippet: Effect of 60 mmHg hydrostatic pressure on ZO-1 in TM cells after 24 h. ZO-1 was immunolabeled with FITC and the nucleus was immunolabeled with hoechst, and observed under a confocal microscope, using identical parameters. The ZO-1 distribution in iHTM is clearly and regular, while that in GTM 3 is irregular and tangled. Cells under 60 mmHg hydrostatic pressure showed significant decreased and irregular distribution of ZO-1 in both iHTM and GTM 3 . The blank group showed that PBS was used instead of mouse antibody against ZO-1, other steps are the same. Every picture involved in the figure is the Z-depth slide that contain most green fluorescence (scale bar=10 µm).

Article Snippet: After blocking the cells with 5% bovine serum albumin (BSA), they were incubated overnight at 4 °C with mouse antibody against ZO-1 (1:50; Invitrogen, Carlsbad, CA) diluted in 1% BSA in PBS.

Techniques: Immunolabeling, Microscopy, Fluorescence

Journal: Scientific Reports

Article Title: Establishment of well-differentiated camelid airway cultures to study Middle East respiratory syndrome coronavirus

doi: 10.1038/s41598-022-13777-y

Figure Lengend Snippet: Establishment and characterization of Bactrian camel and llama AEC cultures. ( A ) Immunofluorescence analysis showing the development of tight-junctions (ZO-1, white) and ciliogenesis (β-tubulin, red) in Bactrian camel and llama AEC cultures over time from 1-day to 4 weeks post ALI exposure. The cells were counterstained with DAPI (blue) to visualize the nuclei. ( B,C ) Ciliogenesis quantification of camel and llama AEC cultures overtime, respectively. Ciliation was quantified by measuring the area above a fluorescence intensity threshold of five random images acquired per condition. ( D ) Transepithelial electrical resistance (TEER) measurement of camel and llama AEC cultures overtime during the differentiation. ( E ) Epithelial morphology of ex vivo tissues (upper panel) and well-differentiated camel and llama AEC cultures (lower panel). ( F ) DPP4 expression in well-differentiated camel and llama AEC cultures, with Vero cells as a positive control. Scale bar is 20 µm.

Article Snippet: Alexa-Fluor ® 647-labelled rabbit anti β-tubulin IV (Cell Signalling Technology, 9F3) and Alexa-Fluor ® 594-labelled mouse antibody against ZO-1 (Thermo Fisher Scientific, 1A12) were used to visualize cilia and tight junctions, respectively.

Techniques: Immunofluorescence, Fluorescence, Ex Vivo, Expressing, Positive Control