For the RPE characterization studies, iPSC-derived RPE was seeded and processed as previously described (19). For the differentiation assay, embryoid bodies were fixed with 4% PFA, permeabilized with 0.1% Triton X-100 and blocked in 10% donkey serum (Millipore) and 1% BSA (Sigma-Aldrich). Primary antibodies were diluted in 1% donkey serum and 1% BSA, and incubated overnight at 4 °C. Secondary antibodies were incubated 45 min at room temperature with 0.2 µg/ml bisBenzimide Hoechst (Sigma-Aldrich) prior to mounting in ProLong Diamond Antifade Mountant (Molecular probes, Life Technologies). Primary antibodies: 1:100 dilution rabbit anti-human ZO-1 (Invitrogen, Life technologies), 1:250 rabbit anti-human LRAT (Abcam), 1:500 mouse anti-human Bestrophin-1 (Abcam), 1:200 mouse anti-Nestin (Novus Biologicals, Lille, France), 1:200 mouse anti-SMA (Dako, Les Ulis, France) and 1:200 mouse anti-AFP (Sigma-Aldrich). Secondary antibodies: 1:500 dilution donkey anti-rabbit IgG-Alexa Fluor 594, and donkey anti-mouse IgG-Alexa Fluor 488 (Jackson ImmunoResearch). Images were taken using a Zeiss ApoTome 2 Upright wide-field microscope (Carl Zeiss SAS).
Rabbit anti human zo 1
Manufactured by Thermo Fisher Scientific
Sourced in United States
Rabbit anti-human ZO-1 is a primary antibody that recognizes the tight junction protein ZO-1 (Zonula Occludens-1) in human samples. It can be used for the detection and analysis of ZO-1 expression in various cell and tissue types.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Mouse anti human fibronectin antibody, by Merck Group (2 mentions)
Superreal premix plus sybr green reagent kit, by Takara Bio (2 mentions)
Mouse anti human e cadherin antibody, by BD (2 mentions)
Alexa fluor 488 donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 594 donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Bisbenzimide hoechst, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Donkey serum, by Merck Group (1 mentions)
Mouse anti sma, by Agilent Technologies (1 mentions)
Mouse anti afp, by Merck Group (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Mouse anti nestin, by Novus Biologicals (1 mentions)
Mouse anti human zo 1, by Thermo Fisher Scientific (1 mentions)
Rabbit anti human cldn5, by Abcam (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Mouse anti human β tubulin, by Merck Group (1 mentions)
Rabbit anti human nlrp3, by Merck Group (1 mentions)
Mouse anti human nlrp3 cryo 2, by Adipogen (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Thermo Fisher Scientific (1 mentions)
9 protocols using rabbit anti human zo 1
1
iPSC-derived RPE Characterization and Differentiation
2
BBB Integrity Markers Quantification
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Markers of BBB integrity were assessed by measuring serum concentrations of S100β, occludin (OCLN), claudin-5 (CLN5), and zonula occludens 1 (Zo-1). To estimate OCLN concentrations, rabbit and mouse anti-human OCLN antibodies (Zymed Labolatories Inc, South San Francisco, CA, USA) were used as capture and detection antibodies, respectively. CLDN5 concentrations were estimated with an in-house enzyme-linked immunosorbent assay (ELISA), using mouse anti-human CLDN5 (Zymed Labolatories Inc.) and rabbit anti-human CLDN5 (Abcam Inc., Cambridge, UK) antibodies for capture and detection, respectively. Zo-1 levels were analysed with rabbit anti-human Zo-1 and mouse anti-human Zo-1 (Invitrogen, Waltham, MA, USA) antibodies for capture and detection, respectively. Goat anti-mouse IgG (H+L)−HRPO was used as the secondary antibody for the OCLN and Zo-1 ELISAs, and goat anti-rabbit IgG (H+L)−HRPO (Invitrogen) was used for the CLDN5 ELISA.
S100β levels were determined using commercially available ELISA kits according to the manufacturer’s instructions (BioVendor Laboratory Medicine Inc., Brno, Czech Republic; and R&D Systems Inc., Minneapolis, MN, USA).
S100β levels were determined using commercially available ELISA kits according to the manufacturer’s instructions (BioVendor Laboratory Medicine Inc., Brno, Czech Republic; and R&D Systems Inc., Minneapolis, MN, USA).
3
Immunofluorescence and Western Blot Analysis of Cellular Markers
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Antibodies were from the following sources: anti-mouse α-smooth muscle actin (Clone 1A4; Sigma-Aldrich), mouse anti-human β-tubulin (Sigma), rabbit anti-human NLRP3 (Sigma), mouse anti-human NLRP3 (Cryo-2, Adipogen), rabbit anti-human Smad 2/3 (Santa Cruz Biotechnology), mouse anti-human KIM-1/TIM-1 (R&D Systems), mouse anti-human E-cadherin (clone 36; BD Biosciences), rabbit anti-human Zo1 (Invitrogen), rabbit anti-human calreticulin (Abcam), rabbit anti-human cytochrome C (Abcam), rabbit anti-human GM130 (Abcam), and rabbit anti-human podocin (Sigma). All primary antibodies were used at a dilution of 1:1000 for immunoblotting and 1:200 for immunofluorescence respectively unless otherwise specified. For mitochondrial labelling, 200 nM of Mitotracker Red CMXRos (Invitrogen) was added to the culture medium and cells incubated for 15 min prior to fixation of cells.
4
Epithelial-Mesenchymal Transition Antibody Panel
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Mouse anti-human E-Cadherin antibody (for western blot) was purchased from BD Bioscience (San Jose, CA, USA). Rabbit anti-human ZO-1, Mouse anti-human α-SMA, FITC-conjugated anti mouse, and FITC-conjugated anti Rabbit antibodies were obtained from Invitrogen (Carlsbad, CA, USA). Rabbit anti-human ZEB1 antibody, Mouse anti-human fibronectin antibody was from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-human SLUG antibody, Rabbit anti-human E-Cadherin antibody (for immunofluorescence) and rabbit anti-human β-actin antibody was purchased from Abcam (Cambridge, MA, USA). Rabbit anti-human Snail antibody was from Santa-Cruze Biotechnology (Santa Cruz, CA, USA). Mouse anti-human Smad2/3, p-Smad2/3, p38, and p-p38 antibodies were from Cell Signaling Technology (Danvers, MA, USA). TRIzol reagent and DAPI (4’,6-diamidino-2-phenylindole) were bought from Invitrogen (Carlsbad, CA, USA). SuperReal PreMix Plus (SYBR Green) reagent kit was from Takara Clontech (Kyoto, Japan). Most of other reagents such as salt and buffer components were analytical grade and obtained from Sigma-Aldrich (St. Louis, MO, USA).
5
Immunofluorescence Staining of Cellular Markers
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The following antibodies and stains were used: Alexa Fluor Rhodamine Phalloidin (Invitrogen Cat.#: R415; 1:50), Hoechst (Invitrogen Cat.#: H3570; 1:10,000), Rat anti-HA (Roche Cat.#: 1186742300; 1:200–1:100), rabbit anti-mouse caspase-3 (R+D systems Cat.#: AF835; 1:100), rat anti-mouse Cd104 (β4-integrin) (BD Biosciences Cat.#: 553745; 1:100), Rat anti-mouse α-18 (gift from Akira Nagafuchi, [25 (link)]; 1:100), Rabbit anti- mouse Olfm4 (Cell Signaling Technology, Cat.#:39141T; 1:400), Rabbit anti- mouse SOX9 (Millipore, Cat.#: AB5535; 1:250), mouse anti- Phospho-Histone H2A.X (Invitrogen, Cat.#: MA1-2022; 1:100), Rabbit anti-mouse non muscle myosin heavy chain IIA (Biolegend, Cat.#: 909001; 1:100), Rabbit anti-mouse non muscle myosin heavy chain IIC (Biolegend, Cat.#: 919201; 1:100), Rat anti-mouse Ki67 (Invitrogen, Cat.# 14-5698-80; 1:100), Rabbit anti-mouse GRASP65 (Abcam, Cat.# ab30315; 1:200), Rat anti-mouse E-cadherin (Invitrogen, Cat.# 13–1900; 1:500), Rabbit anti-Pericentrin (Abcam, Cat.# ab4448; 1:100), Rabbit anti-human ZO-1 (Invitrogen, Cat.# 61–7300; 1:100).
6
Immunofluorescence Analysis of ZO-1 and E-cadherin
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The changes in the expressions of ZO-1 and E-cadherin were analyzed by immunofluorescence analysis. NHNE cells were fixed with 4% paraformaldehyde for 20 min at room temperature (RT) and then treated with 0.1% Triton X-100 for 5 min. Blocking was performed for 30 min at RT with 3% bovine serum albumin in phosphate-buffered saline. Cells were incubated with rabbit anti-human ZO-1 (Invitrogen) or rabbit anti-human E-cadherin (Cell Signaling) antibodies overnight at 4℃ and then incubated with Alexa 488-conjugated goat anti-rabbit IgG (Invitrogen) for 1 hour at RT. Afterwards, the nucleus was stained with DAPI solution (1:1000, Invitrogen) for 1 minute at RT. Between each step, the cells were extensively rinsed three times for 5 minutes each time. Images were taken with a confocal microscope (Olympus FV-1000, Olympus, Tokyo, Japan). All experiments were repeated three times using different batches of NHNE cells.
7
Visualizing E. coli and Bifidobacteria Effects on Tight Junctions
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To visualize the effects of E. coli and bifidobacteria on tight-junction proteins in epithelial layers, cells were treated as described above, but after permeabilization cells were exposed to 5% BSA in PBS for 1 h at room temperature to block non-specific adsorption, washed once with PBS containing 0.1% Triton X-100 (PBST) for 5 min, and subsequently labeled with primary antibody rabbit-anti-human ZO-1 (1:200, #40-2300, Invitrogen) at 4°C overnight. Next, cells were washed twice with PBST for 5 min, and labeled with secondary antibody Rhodamine Red-X Donkey anti-Rabbit (1:100, #711-295-152, Jackson Immunolab, excitation 570 nm/emission 590 nm) for 1 h. Finally, cells were washed with PBST and PBS, each for 5 min and ZO-1 visualized employing fluorescence microscopy with the green laser (Leica DM4000, Germany).
8
Immunostaining of Tight Junction Protein ZO-1
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To assess the expression of tight junction protein ZO-1, ciPTEC-OAT1 cultured on double-coated HFM were fixed with 4% (w/v) paraformaldehyde dissolved in PHEM buffer (120 mM PIPES, 50 mM HEPES, 4 mM MgCl2, 20 mM EGTA) for 15 min. After washing the samples with HBSS, block solution (2% (v/v) FCS, 2% (w/v) bovine serum albumin (BSA), 0.1% (v/v) Tween20 in HBSS) was added. The primary antibody, rabbit anti-human ZO-1 (Invitrogen, Carlsbad, CA, USA), was diluted in blocking buffer (1:200) and incubated overnight at 4 °C. Following three washing steps with HBSS, the secondary antibody, goat anti-rabbit IgG Alexa 568 (Life Technologies, Eugene, OR, USA) was added in a concentration of 1:200 and incubated for 1 h at room temperature. Finally, ProLongTM Gold antifade reagent containing DAPI (Life Technologies, Eugene, OR, USA) was used for nuclear staining, and to mount the fibers containing cells on the Willco glass bottom dishes (WillCo Wells B.V., Amsterdam, The Netherlands). Cells were imaged using confocal microscope (Leica TCS SP8 X, Leica Microsystems CMS GmbH, Wetzlar, Germany) and analyzed using Leica Application Suite X software (Leica Microsystems CMS GmbH).
9
Immunostaining of Cell-Cell Junctions
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Mouse anti-human E-cadherin antibody (category number: 610182) was purchased from BD Biosciences (San Jose, CA, USA). Rabbit anti-human ZO-1 (category number: 61-7300), Alexa Fluor 594-conjugated anti-rabbit antibody, and FITCconjugated anti-mouse antibody were obtained from Invitrogen (Carlsbad, CA, USA). Mouse anti-human fibronectin antibody was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Mouse anti-human connexin 43 antibody (category number: ab11370) and rabbit anti-human b-actin antibody were purchased from Abcam (Cambridge, MA, USA). TRIzol reagent and 4 0 ,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen. The SuperReal PreMix Plus (SYBR Green) reagent kit was obtained from Takara Clontech (Kyoto, Japan). Most of the other reagents, such as salt and buffer components, were of analytical grade and were obtained from Sigma-Aldrich Corp.
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