Suit2-007 cells (metastatic PDAC cell line) were kindly donated by Prof. Malte Buchholz from Philipps-Universität Marburg. Suit2 cells were cultured in Dulbecco’s Modified Eagle’s Media (Merck, Dorset, UK, D8437) supplemented with 10% v/v foetal bovine serum (FBS; Merck, F7524), 2 mM l -glutamine (Merck, G7513), 1% v/v penicillin/streptomycin (Merck, P4333) and 1% v/v fungizone Amphotericin B (Gibco, Thermo Fisher Scientific, UK, 15290-026). Cells were incubated at 37 °C, with 5% CO2. For all RAR-β agonist treatments, cells were exposed to 1 μM RAR-β agonist (CD 2314, Tocris, Abingdon, UK, 3824) 24 h prior to experiments. Gene transfection was performed 48 h prior to experiments, utilising the Neon transfection system (Thermo Fisher Scientific) with 2 μg MLC-2 plasmid (pEGFP-MRLC1, a gift from Tom Egelhoff, Addgene, #35680), 10 μg RAR-β siRNA (Santa Cruz, SC-29466) or 10 μg control siRNA (siRNA-Scr, Santa Cruz, SC-37007). Tissue micro arrays (TMAs) were obtained from Biomax (RAR-β: PA803, MLC-2: PA242e). Primary antibodies used were rabbit anti-RAR-β (Abcam, Cambridge, UK, ab53161), rabbit anti-MLC-2 (Cell Signaling Technology, 3672S), mouse anti-PAN Cytokeratin (Abcam, ab6401), rabbit anti-YAP (Cell Signaling Technology, 4912S) and rabbit anti-laminin (Sigma-Aldrich, L9393). See Supplementary Methods for immunostaining, tissue microarray, ChIP-seq and RT qPCR details.
Mouse anti pan cytokeratin
Manufactured by Abcam
Sourced in United Kingdom, United States, Japan
Mouse anti-PAN Cytokeratin is a primary antibody that recognizes all types of cytokeratin proteins. Cytokeratins are intermediate filament proteins that are expressed in epithelial cells.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Merck Group (1 mentions)
Fungizone amphotericin b, by Thermo Fisher Scientific (1 mentions)
Scr sirna, by Santa Cruz Biotechnology (1 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
Rabbit anti laminin, by Merck Group (1 mentions)
Cd 2314, by Bio-Techne (1 mentions)
Rabbit anti rarβ, by Abcam (1 mentions)
Rabbit anti mlc2, by Cell Signaling Technology (1 mentions)
Rabbit anti yap, by Cell Signaling Technology (1 mentions)
Rabbit anti gfp, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Goat serum, by Vector Laboratories (1 mentions)
Dmi6000b widefield microscope, by Leica (1 mentions)
Nbt bcip ap substrate solution, by Roche (1 mentions)
Mmessage mmachine t7 ultra kit, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Rabbit anti cd31, by Abcam (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Takara Bio (1 mentions)
5 protocols using mouse anti pan cytokeratin
1
Metastatic PDAC Cell Line Characterization
2
Immunofluorescence Analysis of Humanized Mouse Thymus
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Half of the thymic graft from each humanized mouse was fixed in 4% paraformaldehyde for 30 min at room temperature, then transferred to 30% glucose PBS at 4°C overnight. Tissues were then embedded in OCT compound and frozen. Cryosections (5 μm) were blocked with goat serum (Vector Laboratories) for 15 min, and stained with following primary antibodies: rabbit anti-GFP (Millipore/Sigma) and mouse anti-Pan-cytokeratin (Abcam) in room temperature for 3 h or 4°C overnight. Sections were subsequently stained with AF488 rabbit anti-GFP IgG (H+L) antibody (Jackson ImmunoResearch) and AF647 goat anti-mouse IgG (H+L) antibody (Jackson ImmunoResearch) at room temperature for 1 h. All sections were mounted in mounting medium with DAPI (Vector Laboratories). Images were obtained using Leica DMI 6000B wide field microscope.
3
Histological and Molecular Evaluation of Tissue Samples
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Histologic evaluation was done by using 4-μm sections of paraformaldehyde-fixed and paraffin-embedded tissue. Hematoxylin/eosin (H&E) staining was performed according to standard protocols. IHC and ISH experiments were carried out as previously described [22] (link), [25] (link). Primary antibodies used for IHC were mouse anti–proliferating cell nuclear antigen (PCNA, 1:2000), mouse anti–-cytokeratin 19 (CK19, 1:500), and mouse anti-pancytokeratin (1:500) purchased from Abcam (Cambridge, MA). For ISH, riboprobes were generated by PCR amplification with partial cDNA using appropriate primers. Primer sequences used for PCR were 5′-ATGTCTTTCCCCCAGCTGGG-3′ (forward) and 5′-CTAATACGACTCACTATAGGG CTCCAGATCTATCTCCTCTTCG-3′ (reverse) for Irx1a (product size 633 bp), and 5′-ATGTCGTTCCCCCAACTGGG-3′ (forward) and 5′-CTAATACGACTCACTATAGGG CCATATCGATGGTCTCCAGATC-3′ (reverse) for Irx1b (product size 658 bp). The underlined sequences are T7 RNA polymerase-binding sites. In vitro transcription was carried out using mMEssage mMACHINE T7 ultra Kit (Ambion). Hybridization was done at 60°C for overnight, and serial stringent wash was done at 68°C. Hybridized riboprobe was detected by anti-dig antibody binding and detected by NBT/BCIP AP substrate solution (Roche). The slides were counterstained with neutral red.
4
Characterization of Cellular Markers
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Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI-1640 medium, fetal bovine serum (FBS), and penicillin–streptomycin solution were obtained from Invitrogen (Carlsbad, CA, USA). Qiazol was obtained from Qiagen (Valencia, CA, USA), and 2× SYBR Green PCR Master Mix was obtained from Takara Bio (Otsu, Japan). HRP-conjugated anti-β-actin (Santa Cruz Biotechnology Cat# sc-47778HRP, RRID: AB_2714189) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-α-SMA (Abcam Cat# ab5694, RRID: AB_2223021), rabbit anti-CD31 (Abcam Cat# ab28364, RRID: AB_726362), and mouse anti-pan-cytokeratin (Abcam Cat# ab7753, RRID: AB_306047) antibodies were from Abcam (Cambridge, MA, USA). Rabbit anti-COL3A1 antibody (Proteintech Group Cat# 22734-AP-1) was obtained from Proteintech (Rosemont, IL, USA). Rabbit anti-COL6A6 (Thermo Fisher Scientific Cat# PA5-60958, RRID: AB_2640027) antibodies were acquired from Thermo Fisher Scientific, Inc. (Irvine, CA, USA). Rabbit anti-Ku80 (Cell Signaling Technology Cat#2753) antibody was obtained from Cell Signaling Technology (Danvers, MA, USA).
5
Immunofluorescent Analysis of Esophageal Epithelium
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The esophageal stratified epithelial layers were fixed in a 2% neutral formalin solution, and embedded in paraffin. Sections (4 μm thick) were cut and deparaffinized using dimethylbenzene followed by dehydration. After antigen retrieval by autoclaving in HistoVT One (Nacalai Tesque, Kyoto, Japan), Protein Block Serum-Free Ready-to-Use (Dako, Carpinteria, CA, USA) was used to minimize non-specific Ig binding. non-specific labeling was blocked with 5% fetal bovine serum/PBS. Slides were then incubated with primary rabbit anti-IL-33 (MBL, Nagoya, Japan) 1:100 or mouse anti-pan Cytokeratin (Abcam, Cambridge, UK) 1:100 overnight at 4°C, Cy3-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and Alexa 488-conjugated goat anti-mouse IgG (Thermo Fisher Scientific Inc. Waltham, MA) secondary antibodies were used at a 1:1000 dilution for 30 min. Nuclear staining was performed using 4’,6-diamidino-2-phenylindole (DAPI) (Invitrogen). Slides were viewed using a confocal laser-scanning microscope (FV1000; Olympus).
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