LGACC cells were seeded in 8-well chamber slides and cultured until confluent. Cells were then washed with PBS, and fixed for 10 minutes at room temperature with 4% paraformaldehyde (PFA). Cells were permeabilized for 5 minutes with 0.2% Triton X-100, and blocked for 30 minutes with 5% BSA. Cells were then incubated overnight at 4°C with primary antibodies against E-cadherin (250μg/ml, BD Biosciences), PDGFR (dilution 1:200, GeneTex), cytokeratin-5 (dilution 1:200, Abcam), FGFR1 (dilution 10μg/ml, abcam), and the low affinity neurotrophin receptor (p75, dilution 1:100, Abcam). After washing in PBS 3 times, sections were incubated with species-specific fluorescent secondary antibodies for 2 hr at room temperature. Finally, cells were counterstained for filamentous actin (F-actin, dilution 1:200) using AlexaFluor594 phalloidin (dilution 1:300, Thermofisher Scientific), and mounted with Vecta shield (Vector) fluorescent mounting medium containing DAPI. Imaging was performed with a Leica AOBS SP8 confocal microscope (Leica Microsystems Exton, PA, USA).
Cytokeratin 5
Manufactured by Abcam
Sourced in United Kingdom
Cytokeratin 5 is a protein that is part of the cytokeratin family. It is considered a marker for basal cells in various tissues.
Automatically generated - may contain errors
Lab products found in correlation
Mmp 9, by Santa Cruz Biotechnology (2 mentions)
Mmp 2, by Santa Cruz Biotechnology (2 mentions)
Bromodeoxyuridine (brdu), by Thermo Fisher Scientific (2 mentions)
Peroxidase kit, by Vector Laboratories (2 mentions)
Wnt10b h70, by Santa Cruz Biotechnology (2 mentions)
Activated β catenin 8e7, by Merck Group (2 mentions)
Permount, by Thermo Fisher Scientific (2 mentions)
Cytokeratin 14, by Abcam (2 mentions)
F actin, by Thermo Fisher Scientific (1 mentions)
Fgfr1, by Abcam (1 mentions)
Leica aobs sp8 confocal microscope, by Leica Microsystems (1 mentions)
Alexa fluor 594 phalloidin, by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
E cadherin, by BD (1 mentions)
Cytokeratin 10, by Abcam (1 mentions)
Mucin 5ac, by Abcam (1 mentions)
Immu mount, by Thermo Fisher Scientific (1 mentions)
Collagen 4, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
β catenin, by Abcam (1 mentions)
12 protocols using cytokeratin 5
1
Immunofluorescence Staining of LGACC Cells
2
Immunohistochemical Analysis of Tissue Sections
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Paraformaldehyde fixed and paraffin embedded tissues were cut into 5 μm sections, subjected to sodium citrate antigen retrieval, and stained using the M.O.M. Peroxidase kit (Vector Labs, Burlingame, CA) and 3,3′-Diaminobenzidine (DAB). The following antibodies were used: DEK (1:60, BD Biosciences), BrdU (1:100, Molecular Probes, Invitrogen, Grand Island, NY), Wnt10b [H70] (1:50, Santa Cruz), MMP-2 and MMP-9 (1:100 and 1:200, respectively, Santa Cruz), cytokeratin-5 (1:200, Abcam), or activated β-catenin [8E7] (1:100, Millipore). Samples were counterstained with 0.1% Nuclear Fast Red (Poly Scientific, Bay Shore, NY) and preserved with Permount (Fisher Scientific, Pittsburgh, PA). Tissue microarrays were purchased from Imgenex (CBA2 and CBB2; Imgenex, San Diego, CA) for correlations of Ron, DEK, and β-catenin expression in human tissues and scored as previously described.6 (link) For R7 xenograft tumors, staining intensity was quantified by the Threshold tool on Image J. The threshold for each section was set and normalized to each section’s respective total area of tissue as seen at 200x total magnification. Scores were calculated by multiplying the intensity score by the percentage of positive staining cells. The total number of cells counted per section ranged from 122 to 357. For activated β-catenin staining, cells were considered positive if the nucleus showed DAB staining.
3
Immunofluorescent Staining of Tissue Sections
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Frozen sections were stained with primary antibodies to cytokeratin 5 (Abcam, Cat. No.53121), cytokeratin 10 (Abcam, Cat. No. 76318), cytokeratin 14 (Abcam, Cat. No. 7800), collagen IV (Abcam, Cat. No. 6586), β‐catenin (Abcam, Cat. No. 16051), β‐tubulin 4 (Abcam, Cat. No. 11315) and mucin 5AC (Abcam, Cat. No. 3649) followed by staining with species‐specific secondary fluorescent antibodies (Jackson Immuno Research Cat. No. 711546152, 715165150). Finally, samples were counter‐stained with DAPI (Sigma, Cat. No. D9542) and mounted in ImmuMount (Thermo Scientific, Cat. No. 9990402). Data were analysed using a fluorescent Zeiss Imager.D2 microscope software.
4
Immunofluorescence Staining of Neuronal Markers
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Cdk5 (Abcam, Cambridge, UK; clone 2G2), cytokeratin 5 (Abcam, EP1601Y), cytokeratin 8 (BioLegend, London, UK; clone 1E8), drebrin (GeneTex, Isleworth, UK; GTX11068), drebrin (Abcam 176318 & 178408), drebrin (Progen, Heidelberg, Germany; clone Mx823), EB1 (BD Transduction Laboratories, Oxford, UK; clone 5), EB3 (Millipore, Consett, UK; AB6033), p35 (Abcam, ab66064), GAPDH (GeneTex, clone GT239), GFP (Abcam, 6556), laminin (Sigma-Aldrich, Poole, UK; clone LAM-89), p35/25 (Cell Signaling, Hitchin, UK; C64B10), p63 (Abcam, ab735 clone BC4A4), pERK1/2 (Thr202/Tyr204) antibody (Cell Signaling; #9101), pS142-drebrin (Millipore, clone 3C14), tyrosinated α-tubulin (SeroTec, Kidlington, UK; clone YL 1/2), vimentin (Dako, Glostrup, Denmark; clone V9). Horseradish peroxidase-conjugated secondary antibodies were from Dako. Alexa-conjugated secondary antibodies and phalloidin were from Life Technologies, Warrington, UK. BTP2 (YM-58483) was from Cambridge Bioscience (Cambridge, UK; CAY13246). Recombinant human CXCL12 was from PeproTech (London, UK; 300-28A). Matrigel was from Corning (New York, USA; 354234) and fibronectin from Millipore.
5
Comprehensive Lung Tumor Histopathology Analysis
6
Immunohistochemical Analysis of Tissue Sections
7
Western Blot Analysis of Protein Signaling
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Total protein was extracted from HaCaT cells using RIPA lysis buffer (Biosharp, Anhui, China). The total protein was separated using Bis-Tris Gel (ACE Bio, Nanjing, China), transferred onto a polyvinylidene fluoride membrane (Millipore, Burlington, MA), and blocked with 5% nonfat milk in Tris-buffered saline with Tween 20 for 4 hours at room temperature. The membrane subsequently was incubated with the primary antibody at 4°C overnight. The following antibodies were used: Mek1/2, phospho-Mek1/2 (Ser221), Erk1/2, phospho-Erk1/2 (Thr202/Tyr204), NF-κB (P65) (1:1000; Cell Signaling Technology, Danvers, MA), phospho-P65 (Ser536) (1:1000; Santa Cruz Biotechnology, Dallas, TX), LMO4, involucrin (1:1000; ZENBIO, Chengdu, China), and cytokeratin 1 and cytokeratin 5 (1:2000; Abcam, Cambridge, MA). The following day, the membranes were incubated with the secondary antibody goat anti-rabbit IgG or horse anti-mouse IgG (1:5000; Cell Signaling Technology) at room temperature for 2 hours. Finally, chemiluminescence was detected using WesternBright ECL horseradish peroxidase substrate (Advansta, San Jose, CA). The relative protein quantification analysis was performed using ImageJ Fiji software version 2.15.0 (https://imagej.net/software/fiji).
8
Kidney Tissue Immunostaining Protocol
9
Histone H3 and Cytokeratin 5 Citrullination
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100 µg ml−1 histone H3 (Abcam) or 20 µg ml−1 cytokeratin 5 (Abcam) was incubated with 200 nM PAD6 or 100 nM PAD4 (Cayman) ± 50 µM GSK484 (Sigma) in citrullination buffer (50 mM Tris–HCl pH 7.6, 1 mM CaCl2, 200 mM NaCl2, 2 mM DTT) in a 384-well Nunc MaxiSorp plate (Thermo Scientific) for 3 h at 37°C and 300 r.p.m. shaking. The reaction was stopped by the addition of EGTA, pH 8.0, to a final concentration of 50 mM. The citrullination was detected using the alkyne-biotin based method as described by Hensen et al. (2015 ▸ ). The blots were scanned using the LiCor Odyssey CLx and quantification of protein bands was performed with the integrated Image Studio software.
10
Comprehensive Immunohistochemistry Protocol for Tumor Cell Profiling
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Primary antibodies: Cytokeratin 19 (Dako, Clone RCK108), Cytokeratin 14 (Abcam, Clone LL002), Vimentin (Cell Signaling, Clone D21H3), Cytokeratin 5 (Abcam, Clone EP1601Y), Cytokeratin 17 (Thermo, clone E3), Claudin 4 (R&D Systems, Clone 382321), Cytokeratin 8 (K8, Abcam, Clone M20), Ku80 (Cell Signaling, Clone C48E7), Ki67 (DAKO, Clone MIB-1), GATA3 (Cell Signaling, Clone D13C9), Cytokeratin 18 (Cell Signaling, Clone DC10).
Secondary antibodies: (all LifeTech unless noted); goat-anti-mouseIgG1-Alexa647, goat-anti-mouse IgG3-Alexa488, goat-anti-mouseIgG2a-Alexa488, goat-anti-mouseIgG2b-Alexa488, donkey-anti-rabbit-Alexa568, donkey-anti-goat-Alexa647, goat-anti-rabbit-dylight755 (Thermo).
Small molecule inhibitors: All drugs, unless otherwise noted, were purchased from Selleckchem including (+)-JQ1 for in vitro experiments. BEZ235 was purchased from LC Laboratories and (+)-JQ1 for in vivo studies was provided by Jay Bradner at the Dana-Farber Institute of Harvard, Cambridge, MA. All in vitro inhibitor stocks were solubilized in DMSO and stored as 10 mM stock solutions at −80 °C.
Secondary antibodies: (all LifeTech unless noted); goat-anti-mouseIgG1-Alexa647, goat-anti-mouse IgG3-Alexa488, goat-anti-mouseIgG2a-Alexa488, goat-anti-mouseIgG2b-Alexa488, donkey-anti-rabbit-Alexa568, donkey-anti-goat-Alexa647, goat-anti-rabbit-dylight755 (Thermo).
Small molecule inhibitors: All drugs, unless otherwise noted, were purchased from Selleckchem including (+)-JQ1 for in vitro experiments. BEZ235 was purchased from LC Laboratories and (+)-JQ1 for in vivo studies was provided by Jay Bradner at the Dana-Farber Institute of Harvard, Cambridge, MA. All in vitro inhibitor stocks were solubilized in DMSO and stored as 10 mM stock solutions at −80 °C.
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