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Keratin 6

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Keratin 6 is a structural protein found in hair, nails, and skin. It is an essential component of the cytoskeleton in epithelial cells. Keratin 6 provides strength, flexibility, and protection to these tissues.

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Lab products found in correlation

Keratin 10, by Fortrea (3 mentions) Phospho stat3 tyr705, by Cell Signaling Technology (2 mentions) Ha tag, by Cell Signaling Technology (2 mentions) F4 80, by Bio-Rad (2 mentions) Vectastain abc peroxidase kit, by Vector Laboratories (2 mentions) 3 3 diaminobenzidine substrate, by Merck Group (2 mentions) Occludin, by BD (1 mentions) Keratin 76, by Merck Group (1 mentions) Phospho histone h3, by Cell Signaling Technology (1 mentions) E cadherin, by Thermo Fisher Scientific (1 mentions) Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (1 mentions) Claudin 1, by Abcam (1 mentions) Alexa 594, by Thermo Fisher Scientific (1 mentions) Dp72 microscope, by Olympus (1 mentions) Hoechst, by Thermo Fisher Scientific (1 mentions) Anti cd45, by BD (1 mentions) Cellsens standard software, by Olympus (1 mentions) Trp 2, by Abcam (1 mentions) Envision system hrp, by Agilent Technologies (1 mentions) Fontana masson stain kit, by Merck Group (1 mentions)

10 protocols using keratin 6

1

Comprehensive Histological Analysis of Skin Tissue

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Fixed skin tissues were embedded in paraffin and sectioned. The sections
were stained with hematoxylin & eosin, hematoxylin alone, or with
hematoxylin combined with the following antibodies: BrdU, total p53,
γ-H2AX [14 (link)]; Keratin-6 (rabbit
polyclonal, 1:500, Covance), Keratin-10 (rabbit polyclonal, 1:500, Covance), or
TRP2 (rabbit polyclonal, 1:500, Abcam). The tissues were then labeled with
secondary antibodies: Envision+ System HRP labeled polymer anti-rabbit HRP
(Dako) was used for the rabbit polyclonal antibodies. Melanin was stained with a
Fontana-Masson stain kit (Sigma-Aldrich Procedure No. HT200). Stained slides
were digitally scanned using a ScanScope CS (Aperio).
Lange S.S., Bhetawal S., Reh S., Powell K.L., Kusewitt D.F, & Wood R.D. (2018). DNA polymerase ζ deficiency causes impaired wound healing and stress-induced skin pigmentation. Life Science Alliance, 1(3), e201800048.
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2

Immunohistochemical Analysis of Phospho-STAT3

Cited 1 time
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Immunohistochemical analyses on paraffin sections were performed as described (Lin et al., 2010 (link)). The antigen retrieval method and dilutions of primary antibodies used were as follows: EDTA and 1:50 for phospho-STAT3 (Tyr705); Trilogy and 1:50 for HA tag (both antibodies from Cell Signaling Technology, Beverly, MA); and 1:1,000 for keratin 6 (Covance, Princeton, NJ). Sections were counterstained with hematoxylin and mounted.
Lin M.H, & Miner J.H. (2014). Fatty Acid Transport Protein 1 can compensate for Fatty Acid Transport Protein 4 in the developing mouse epidermis. The Journal of investigative dermatology, 135(2), 462-470.
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3

Immunofluorescence Staining of Tight Junctions

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Staining for LacZ expression was performed as previously described [47] on frozen sections and counterstained with Nile Red. Immunofluorescence experiments were performed after citrate based antigen retrieval. Primary antibodies were ZO-1 (Invitrogen cat# 339100), Occludin (BD Transduction cat# 611090), Claudin-1 (ABCAM cat# ab15098), cytokeratin14 - LLOO2 (ABCAM cat # ab7800), keratin10 (Covance PRB-159P), keratin6 (Covance cat # PRB-169P), Ecadherin (Life Technologies, 13-1900), phospho-histone H3 (Cell Signalling, #9708), PCNA (Santa Cruz Biotechnology sc-9857), CLDN1 (Santa Cruz Biotechnology, sc-81796), Keratin 76 (Sigma-Aldrich HPA019696) and Keratin 76 (Sigma-Aldrich HPA019656), Filaggrin (FLG- Covance PRB-417P), FASN (Santa Cruz Biotechnology, sc-48357), and Melan-A (MEL-A, Santa Cruz Biotechnology, sc-20032). All secondary antibodies were AlexaFluor conjugated (Invitrogen).
DiTommaso T., Cottle D.L., Pearson H.B., Schlüter H., Kaur P., Humbert P.O, & Smyth I.M. (2014). Keratin 76 Is Required for Tight Junction Function and Maintenance of the Skin Barrier. PLoS Genetics, 10(10), e1004706.
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4

Skin Histology and Immunostaining Protocol

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Skin samples were fixed in 10% neutral buffered formalin, paraffin-embedded, and sectioned for routine histology staining (H&E). For skin immunofluorescence, parafin sections were de-waxed, subjected to heat-induced epitope retrieval (HIER) with citrate buffer, blocked in 3% goat serum, then permeabilised with 0.3% Triton X-100, and stained with keratin 6 (Covance) or keratin 14, and goat anti-rabbit Alexa 594 (Invitrogen, Carlsbad, CA) antibodies. Nuclei were visualized using Hoechst (Invitrogen). For tissue immunohistochemistry, sections were dewaxed and subjected to antigen retrieval in trypsin buffer or HIER with citrate buffer and stained with anti-CC3 (9691; Cell Signaling Technology, Danvers, MA), anti-CD45 (BD, San Jose, CA) and goat anti-rabbit biotinylated antibodies. Images were taken using a DP72 microscope and cellSens Standard software (Olympus, Tokyo, Japan).
Etemadi N., Chopin M., Anderton H., Tanzer M.C., Rickard J.A., Abeysekera W., Hall C., Spall S.K., Wang B., Xiong Y., Hla T., Pitson S.M., Bonder C.S., Wong W.W., Ernst M., Smyth G.K., Vaux D.L., Nutt S.L., Nachbur U, & Silke J. (2015). TRAF2 regulates TNF and NF-κB signalling to suppress apoptosis and skin inflammation independently of Sphingosine kinase 1. eLife, 4, e10592.
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5

Immunohistochemical Analysis of Phospho-STAT3

Cited 1 time
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Immunohistochemical analyses on paraffin sections were performed as described (Lin et al., 2010 (link)). The antigen retrieval method and dilutions of primary antibodies used were as follows: EDTA and 1:50 for phospho-STAT3 (Tyr705); Trilogy and 1:50 for HA tag (both antibodies from Cell Signaling Technology, Beverly, MA); and 1:1,000 for keratin 6 (Covance, Princeton, NJ). Sections were counterstained with hematoxylin and mounted.
Lin M.H, & Miner J.H. (2014). Fatty Acid Transport Protein 1 can compensate for Fatty Acid Transport Protein 4 in the developing mouse epidermis. The Journal of investigative dermatology, 135(2), 462-470.
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6

Histological Analysis of Skin Tissue

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Fixed skin tissues were embedded in paraffin and sectioned. The sections were stained with hematoxylin and eosin, hematoxylin alone, or with hematoxylin combined with the following antibodies: BrdU, total p53, and γ-H2AX (14 (link)); keratin-6 (rabbit polyclonal, 1:500; Covance); keratin-10 (rabbit polyclonal, 1:500; Covance); or TRP2 (rabbit polyclonal, 1:500; Abcam). The tissues were then labeled with secondary antibodies.EnVision+ System HRP labeled polymer; anti-rabbit HRP (Dako) was used for the rabbit polyclonal antibodies. Melanin was stained with a Fontana-Masson stain kit (Sigma-Aldrich procedure no.: HT200). Stained slides were digitally scanned using a ScanScope CS (Aperio).
Lange S.S., Bhetawal S., Reh S., Powell K.L., Kusewitt D.F, & Wood R.D. (2018). DNA polymerase ζ deficiency causes impaired wound healing and stress-induced skin pigmentation. Life Science Alliance, 1(3), e201800048.
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7

Comprehensive Immunohistochemical Analysis

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Ki67 (1:500, Novocastra), Keratin 10, Keratin 6, Loricrin (1:1,000, Covance), p53 (1:50, NCL‐p53‐CM5p, Novocastra), cleaved‐caspase 3 (1:200, CC3, cs‐96615, Cell Signaling), PCNA (1:200, DAKO), DNMT1 (1:200, Abcam, ab188453), CGAS (1:100 Cell Signaling, 31659; 1:200 Cell Signaling 15102) 5 mC (1:500, Diagenode 006‐500), CD45 (1:1,000, Abcam ab10558), and histone H3S10ph (1:2,000, Santa Cruz Biotechnology SC‐8656‐R).
Beck M.A., Fischer H., Grabner L.M., Groffics T., Winter M., Tangermann S., Meischel T., Zaussinger‐Haas B., Wagner P., Fischer C., Folie C., Arand J., Schöfer C., Ramsahoye B., Lagger S., Machat G., Eisenwort G., Schneider S., Podhornik A., Kothmayer M., Reichart U., Glösmann M., Tamir I., Mildner M., Sheibani‐Tezerji R., Kenner L., Petzelbauer P., Egger G., Sibilia M., Ablasser A, & Seiser C. (2021). DNA hypomethylation leads to cGAS‐induced autoinflammation in the epidermis. The EMBO Journal, 40(22), e108234.
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8

Histopathological Assessment of Skin and Tumor Tissues

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Skin and tumor tissues were fixed for 24 h in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS), processed into paraffin, sectioned into sagittal 3-µm sections and stained with hematoxylin and eosin, Masson's trichrome or toluidine blue prior to histopathological assessment. Immunohistochemical analysis was performed as described previously (51 (link)). Antibodies recognizing keratin-6 (Covance, Chantilly, VA), and F4/80 (AbD Serotec, Raleigh, NC) were used for staining. Epithelial cell proliferation was visualized by intraperitoneal injection of 10 µg/g of 5-bromo-2′-deoxyuridine (BrdU), 2 h prior to euthanasia. BrdU incorporation was detected with a mouse anti-BrdU antibody (Accurate, Westbury, NY). Bound antibodies were visualized with the Vectastain ABC peroxidase kit as recommended by the manufacturer (Vector Laboratories, Burlingame, CA) and developed with the addition of the 3,3’-diaminobenzidine substrate (Sigma, St Louis, MO). Slides were digitalized using a ScanScope (Aperio, Vista, CA) and the height of the epidermis (epithelial thickness), mast cell count per 105 µm2 and the number of BrdU positive basal cells (basal proliferation rates) were quantified.
Sales K.U., Friis S., Konkel J.E., Godiksen S., Hatakeyama M., Hansen K.K., Rogatto S.R., Szabo R., Vogel L.K., Chen W., Gutkind J.S, & Bugge T.H. (2014). Non-hematopoietic PAR-2 is essential for matriptase-driven pre-malignant progression and potentiation of ras-mediated squamous cell carcinogenesis. Oncogene, 34(3), 346-356.
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9

Histopathological Analysis of Tumor Sections

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Five micron serial tumor sections were deparaffinized, gradually hydrated, and stained for hematoxylin and eosin (H&E) and then examined microscopically. 20 MMTV-CD8-IGF1R tumors and 22 MMTV-ErbB2 control tumors were examined by H&E. Tumors were stained by immunohistochemistry with Troma1-Keratin 8 (1:200; Developmental Studies Hybridoma Bank), Keratin 14 (1:200; Covance), Keratin 6 (1:200, Covance). Trichome staining was performed using the Masson Trichome Stain kit (Sigma, HT15–1KT) according to manufacturer’s instructions.
Farabaugh S.M., Litzenburger B.C., Elangovan A., Pecar G., Walheim L., Atkinson J.M, & Lee A.V. (2020). IGF1R Constitutive Activation Expands Luminal Progenitors and Influences Lineage Differentiation During Breast Tumorigenesis. Developmental biology, 463(1), 77-87.
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10

Histopathological Assessment of Skin and Tumor Tissues

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Skin and tumor tissues were fixed for 24 h in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS), processed into paraffin, sectioned into sagittal 3-µm sections and stained with hematoxylin and eosin, Masson's trichrome or toluidine blue prior to histopathological assessment. Immunohistochemical analysis was performed as described previously (51 (link)). Antibodies recognizing keratin-6 (Covance, Chantilly, VA), and F4/80 (AbD Serotec, Raleigh, NC) were used for staining. Epithelial cell proliferation was visualized by intraperitoneal injection of 10 µg/g of 5-bromo-2′-deoxyuridine (BrdU), 2 h prior to euthanasia. BrdU incorporation was detected with a mouse anti-BrdU antibody (Accurate, Westbury, NY). Bound antibodies were visualized with the Vectastain ABC peroxidase kit as recommended by the manufacturer (Vector Laboratories, Burlingame, CA) and developed with the addition of the 3,3’-diaminobenzidine substrate (Sigma, St Louis, MO). Slides were digitalized using a ScanScope (Aperio, Vista, CA) and the height of the epidermis (epithelial thickness), mast cell count per 105 µm2 and the number of BrdU positive basal cells (basal proliferation rates) were quantified.
Sales K.U., Friis S., Konkel J.E., Godiksen S., Hatakeyama M., Hansen K.K., Rogatto S.R., Szabo R., Vogel L.K., Chen W., Gutkind J.S, & Bugge T.H. (2014). Non-hematopoietic PAR-2 is essential for matriptase-driven pre-malignant progression and potentiation of ras-mediated squamous cell carcinogenesis. Oncogene, 34(3), 346-356.
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