5 × 106 YTS-CLEC16A NK cells and ex vivo NK cells were used in Qproteome Cell Compartment Kit according to the manufacturer's instructions (Qiagen). Ten micrograms of protein per fraction was used for western blot. The membranes were probed for CLEC16A (Abgent), Rab5, Rab7, GAPDH, Tim-23, Histone H3, ERp72, Syntaxin 6 (Cell Signaling), and Cytokeratin 18 (Abcam).
Cytokeratin 18
Manufactured by Abcam
Sourced in United States
Cytokeratin 18 is a type I intermediate filament protein that is specifically expressed in simple and single-layered epithelial cells. It is commonly used as a marker for the identification and characterization of epithelial cells.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Cell Signaling Technology (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Cytokeratin 4, by Abcam (2 mentions)
Vimentin, by Abcam (2 mentions)
Cytokeratin 8, by Abcam (2 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
Syntaxin 6, by Cell Signaling Technology (1 mentions)
Clec16a, by Abcepta (1 mentions)
Erp72, by Cell Signaling Technology (1 mentions)
Qproteome cell compartment kit, by Qiagen (1 mentions)
Alexa fluor 647 conjugated goat anti rabbit igg, by Abcam (1 mentions)
Eclipse ti2 series, by Nikon (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Paraformaldehyde (pfa), by Solarbio (1 mentions)
Cytokeratin 14, by Abcam (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Abcam (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Peroxidase blocking solution, by Agilent Technologies (1 mentions)
Paraformaldehyde (pfa), by Muto Pure Chemicals (1 mentions)
13 protocols using cytokeratin 18
1
NK Cell Compartment Proteome Analysis
2
Lipofuscin Characterization in Liver Cells
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The presence of lipofuscin, a highly oxidized insoluble protein that accumulates in the cytoplasm, was identified using Sudan Black B (SBB) and Fontana Masson staining only at 6 months of age. Pictures were quantified using ImageJ software, and a “stack image” and a color threshold were applied to identify the stained structure. Results are reported as a red stained area percentage among the total area [30 (link),42 (link)]. Additionally, the autofluorescence of liver lipofuscin was detected using an inverted fluorescent microscope (Eclipse Ti2 Series-Nikon) at birth and 6 months of age. Lipofuscin exhibits broad-spectrum autofluorescence [43 (link),44 (link)]. A GFP filter set was applied with an exposure time of 400 ms throughout the observation. Pictures were taken by a single examiner (C.Y.).
To localize lipofuscin deposits at 6 months of age, hepatocytes were stained with cytokeratin 18 (1/100, Abcam) overnight at 4 °C. Sections were then washed with PBS and incubated for two hours with Alexa Fluor-647-conjugated goat anti-rabbit IgG (1/200, Abcam). Sections were then rinsed with PBS and mounted using Fluoromount g mounting medium with DAPI. A negative control was established through incubation only with secondary antibody. Slides were observed blindly using a fluorescence microscope (Nikon, Eclipse Ti2 Series) by the same experimenter (C.Y.).
To localize lipofuscin deposits at 6 months of age, hepatocytes were stained with cytokeratin 18 (1/100, Abcam) overnight at 4 °C. Sections were then washed with PBS and incubated for two hours with Alexa Fluor-647-conjugated goat anti-rabbit IgG (1/200, Abcam). Sections were then rinsed with PBS and mounted using Fluoromount g mounting medium with DAPI. A negative control was established through incubation only with secondary antibody. Slides were observed blindly using a fluorescence microscope (Nikon, Eclipse Ti2 Series) by the same experimenter (C.Y.).
3
Immunofluorescence Analysis of Cytoskeletal Proteins
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Cells were fixed for 30 min in 4% paraformaldehyde (Solarbio, Beijing, China), permeabilized for 15 min in 0.2% Triton X-100 (Sigma, St. Louis, Mosby, USA), incubated for 2 h in blocking buffer [3% bovine serum albumin (BSA) in PBS], and treated with primary antibody against cytokeratin 18 (1:100, Abcam, Cambridge, Massachusetts, USA), cytokeratin 14 (1:100, Abcam, Cambridge, Massachusetts, USA), and NPR-B (1:100, Bioss, Beijing, China) overnight at 4°C. The secondary antibody used was goat antirabbit IgG-Alexa Fluor® 488 (1:100, Abcam, Cambridge, Massachusetts, USA). The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime, Shanghai, China) for 5 min at room temperature. Images were captured using a fluorescence microscope (Olympus, Tokyo, Japan).
4
Characterization of Human Mucosal Tissues
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Harvested nasal mucosal cell sheets were fixed using 4% paraformaldehyde (Muto Pure Chemicals, Tokyo, Japan). After dehydration, the samples were embedded in paraffin. Human native nasal mucosal tissue, middle ear mucosal tissue, and oral mucosal tissue were obtained via endoscopic sinus surgery, mastoidectomy and tonsillectomy, respectively. These specimens were also embedded in paraffin. Paraffin embedded samples were cut into 4-μm-thick sections and placed on glass slides. The sections were deparaffinized by xylene and ethanol, and then washed with pure water. The sections were stained with hematoxylin and eosin (HE staining). For immunohistochemistry, deparaffinized sections were heated with citrate (Dako) to uncover antigens. Next, the sections were incubated with a peroxidase blocking solution (Dako) and a protein blocking solution (Nacalai tesque) to prevent non-specific reactions. The sections were then incubated with cytokeratin 4 (Abcam), cytokeratin 8 (Santa Cruz, CA, USA) and cytokeratin 18 (Abcam), diluted in blocking buffer at 4 °C overnight. Following primary antibody reaction, sections were washed thrice with PBS, 5 min each turn. Samples were then allowed to react with secondary antibodies and HRP polymer (Dako) at room temperature for 1 h. The sections were washed again and covered with 3,3′-diaminobenzidine (Dako). Nuclei were stained using hematoxylin.
5
Immunofluorescence Analysis of Skin Tissue
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First, the skin tissue samples were fixed in 4% paraformaldehyde (Merck, Germany) for 3 h, at room temperature. Next, the samples were washed with PBS and processed for paraffin embedment. After sectioning on four separate glass slides and deparaffinized, Rehydrated samples were blocked with 3% BSA for 1 h and then primary antibodies [1:100 diluted], VIMENTIN (Abcam), E-CADHERIN (Abcam), PANCYTOKERATIN (zymed) and CYTOKERATIN 18 (Abcam) were used in a humidified chamber at 4 °C overnight. The samples were washed with PBS containing 0.025% tween 20 (PBST) and they were incubated with secondary antibody Alexa Fluor 555 (goat anti-mouse, Invitrogen) for 1 h. The samples were washed with PBST and stained with DAPI for 5 min. Finally, immunofluorescence images were captured using fluorescent microscope (BioTek, USA) on days 0, 1, and 7.
6
Protein Extraction and Western Blot Analysis
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Cells were lysed to extract the entire protein contained in a radioimmunoprecipitation assay buffer [50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS] containing 1×protease inhibitor buffer. Protein concentrations were determined using a BCA kit (Thermo Scientific, Shanghai, China). Equal amounts of protein lysates were fractionated by SDS-PAGE and transferred to nitrocellulose membranes (PALL, Shanghai, China). The membranes were blocked with 5% skimmed milk and then incubated with gentle shaking overnight at 4°C, with the primary antibody plus 5% bovine serum albumin in Tris-buffered saline with Tween (TBS-T: 10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20). The following primary antibodies are β-actin, GAPDH (1: 1,000; Cell Signaling Technology, Shanghai, China), cytokeratin 18 (1: 1,000; Abcam, Shanghai, China), and SV40T (1: 100; Santa Cruze, Shanghai, China). The horseradish peroxidase (HRP)-conjugated secondary antibodies are goat anti-rabbit IgG and horse anti-mouse IgG (1: 5,000; Cell Signaling Technology). The target bands were detected using the Pierce ECL Plus Western Blotting Substrate (Thermo Scientific).
7
Immunocytochemical Analysis of Protein Markers
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The expression of protein markers (cytokeratin 8, cytokeratin 18, and vimentin) in YMECs was determined by immunocytochemical analysis. Cells were seeded in 12-well culture plates at a density of 2×104 cells/well and cultured until they reached confluence. The cells were washed with DPBS, fixed with 4% paraformaldehyde for 30 min, and treated with 1% Triton X-100 for 5 min. The cells were subsequently blocked at 37°C for 1 h in 1% bovine serum albumin (BSA). The slides were incubated overnight with the primary antibodies for cytokeratin 8 (Abcam, USA), cytokeratin 18 (Abcam, USA), and vimentin (Abcam, USA), which were diluted to1∶50, 1∶100, and 1∶200, respectively. The cells were washed three times for 5 min each with DPBS and then incubated with secondary antibodies, namely, FITC-conjugated monoclonal goat anti-mouse IgG (Beyotime, China) and Cy3-conjugated monoclonal goat anti-mouse IgG (Beyotime, China), for 1 h in the dark. Hoechst 33342 was used as a nuclear counterstain. Lastly, the slides were washed three times and visualized with a fluorescent phase-contrast microscope (Olympus, DP 70, Japan).
8
Immunofluorescence Analysis of Liver Tissue
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Frozen sections of liver tissue were prepared immediately after sampling. The sections were fixed with ice-cold acetone for 15 min, sealed with 10% goat serum at 4 °C for 30 min, and permeabilized with 0.2% Triton X-100 for 15 min. This was followed by incubation of the sections with antibodies against cytokeratin 18 (1:100, Abcam, USA) or F4/80 (1:250, CST, USA) at 4 °C overnight. The sections were incubated with Cy3-conjugated secondary antibody (Beyotime, 1:400, China) and dyeing with TUNEL and DAPI (Beyotime, China). Changes in cellular calcium ion concentration were analyzed via immunofluorescence staining. Briefly, after macrophages were treated with LPS and AuNPs, the cells were incubated with Fura-4 AM (MCE, USA) and counterstained with DAPI (Beyotime). Fluorescence images were captured using a ZEISS 780 laser confocal microscope (Zeiss, Germany).
9
Immunofluorescence Staining of Mouse Mammary Glands
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Paraffin embedded histological sections of mouse mammary glands were used for immunofluorescence staining. The slides were deparaffinised and hydrated by washing with xylene (twice), 100 % EtOH, 90 % EtOH, 70 % EtOH and water for 5 mins. Antigen retrieval was carried out by boiling the slides for 15–20 min in citrate buffer and allowed to cool at room temperature for 15 min. After incubation in water the slides were permeabilized with 0.5 % Triton X-100. After two PBS washes, sections were blocked with 10 % goat serum for 2 h. Then primary antibody was added to the slides and incubated overnight at 4 °C in a humidified chamber. Primary antibodies used were cytokeratin 18 (Abcam, at 1:300 dilution), and cytokeratin 14 (Covance, CA; 1:400), cleaved caspase -3 (Cell Signaling Technology, 1:200), Ki67 (Abcam, 1:200). The expression of each protein was detected using either FITC, PE or Cy3 conjugated secondary antibodies (at 1:300 dilution). DAPI (Sigma, USA) or Topro-3 (5 μM, Molecular Probes, Eugene, OR) were used to stain the nculeus.
10
Human Urothelial Cell Culture and Differentiation
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Dulbecco’s Modified Eagle’s Medium DMEM (GIBCO, Waltham, MA, USA), collagenase type l (Worthington, Lakewood, NJ, USA), FBS (GIBCO, Waltham, MA, USA), streptomycin and penicillin and 20 mM L-glutamine (Euroclone, Italy), 0.25% trypsin–0.04% EDTA (GIBCO, Waltham, MA, USA), (SV-HUC-1) ATCC (CRL-9520), StemPro Adipogenesis differentiation media (Invitrogen, Hercules, CA, USA), Trizol reagent (Invitrogen, Hercules, CA, USA), Human MSC Analysis Kit (BD, Franklin Lakes, NJ, USA), iScript reverse transcription supermix (BioRad, Hercules, CA, USA), iQTM SYBR mix (BioRad, Hercules, CA, USA), cytokeratin-18 (Abcam, ab668, 1:200), uroplakin-2 (Santa Cruz, sc-15178, 1:50), ELISA kits (Abcam, UK).
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