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Keratin 5

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Keratin 5 is a cytoskeletal protein found in basal cells of stratified squamous epithelia. It is a structural component of the intermediate filament system.

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Lab products found in correlation

Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Antigen unmarking solution, by Vector Laboratories (1 mentions) Cytokeratin 8, by BioLegend (1 mentions) Pannoramic 250 flash 3, by 3DHISTECH (1 mentions) Il 6r, by R&D Systems (1 mentions) Tunel assay kit, by Abcam (1 mentions) Pannoramic viewer software, by 3DHISTECH (1 mentions) Immpact dab kit, by Vector Laboratories (1 mentions) P stat5, by Cell Signaling Technology (1 mentions) Mayer s hematoxylin, by Merck Group (1 mentions) Foxc1, by Santa Cruz Biotechnology (1 mentions) Vectastain abc system, by Vector Laboratories (1 mentions) Myc tag, by Cell Signaling Technology (1 mentions) Vectastain abc kit, by Vector Laboratories (1 mentions) Bromodeoxyuridine (brdu), by Santa Cruz Biotechnology (1 mentions) A1 confocal microscope, by Nikon (1 mentions) Nis element, by Nikon (1 mentions) Cd11b, by BD (1 mentions) Alexa fluor 488 and 594, by Thermo Fisher Scientific (1 mentions) Evos fl auto cell imaging system, by Thermo Fisher Scientific (1 mentions)

7 protocols using keratin 5

1

Comprehensive Immunohistochemistry Profiling

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Immunohistochemistry was performed as previously described (17 (link)). A pressure cooker (95°C for 30 min followed by 120°C for 10 s) was used for antigen retrieval using Antigen Unmarking Solution (Vector Laboratories). Antibodies specific to CHD1 (Sigma, #HPA022236), AKT-473P (Cell Signaling, #4060s), Keratin 5 (BioLegend 905501), Cytokeratin-8 (BioLegend 904801), CD8 (Bioss bs-0648R), Ly6G (BioLegend 127601), CD15 (DAKO M3631), IL‑6R (R&D Systems AF1830-SP) and Ki67 (Thermo Scientific RM 9106-S1) were purchased. TUNEL staining was performed using the TUNEL Assay Kit (Abcam ab206386) according to manufacturer’s instructions. Slides were scanned using Pannoramic 250 Flash III (3DHISTECH Ltd) and images were captured through Pannoramic Viewer software (3DHISTECH Ltd). Human prostate hyperplasia and cancer tissue array samples were purchased from US Biomax (PR753a).
Zhao D., Cai L., Lu X., Liang X., Li J., Chen P., Ittmann M., Shang X., Jiang S., Li H., Meng C., Flores I., Song J.H., Horner J.W., Lan Z., Wu C.J., Li J., Chang Q., Chen K.C., Wang G., Deng P., Spring D.J., Wang Y.A, & DePinho R.A. (2020). Chromatin Regulator, CHD1, Remodels the Immunosuppressive Tumor Microenvironment in PTEN-Deficient Prostate Cancer. Cancer discovery, 10(9), 1374-1387.
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2

Immunohistochemical Profiling of Murine Mammary Glands

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The #4 mammary glands from wildtype control mice and MMTV-FOXC1 transgenic mice were fixed in formalin (10%) and paraffin-embedded by the Cedars-Sinai Medical Center pathology lab. Sectioning and H&E staining were also performed. Immunohistochemistry was conducted using Vectastain ABC Kit (Vector laboratories) and ImmPACT DAB Kit (Vector). Sections were deparaffinized and rehydrated by xylene and gradient ethanol, respectively. An antigen retrieval method using microwave pretreatment and 0.01 M sodium citrate buffer (pH6) was used for all antibodies (Vector laboratories, Burlingame, CA). Primary antibodies were FOXC1 (1:100, sc-21394, Santa Cruz), Myc-Tag (1:100, #2272, Cell Signaling), ER (1:100, sc-542 Santa Cruz), PR (1:100, sc-538 Santa Cruz), Ki67 (1:100, 790–4286, Roche Diagnostics), Elf5 (1:100, sc-9645, Santa Cruz), Keratin 5 (1:100, 905501, Biolegend), Keratin 8 (1;100, DSHB), p-Stat5(1:100,9359 s, Cell Signaling). The signal was visualized using the VECTASTAIN ABC Systems (Vector laboratories). Counterstaining was performed with Mayer’s hematoxylin (Sigma). The percentages were calculated by comparing stained nuclei to total nuclei in epithelial cells.
Gao B., Qu Y., Han B., Nagaoka Y., Katsumata M., Deng N., Bose S., Jin L., Giuliano A.E, & Cui X. (2017). Inhibition of lobuloalveolar development by FOXC1 overexpression in the mouse mammary gland. Scientific Reports, 7, 14017.
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3

In-vivo BrdU Proliferation Assay

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For in-vivo assessment of cell proliferation, animals were injected intraperitoneally with 100μg/g body weight BrdU 24hrs, 4hrs, and 1hr before euthanasia. Immunohistochemical analysis was performed as we previously described5 (link). Antibodies against Keratin 5 (Biolegend), CD31 (Abcam), CD11b (BD Biosciences), Ly6G (BD Biosciences), IBA1 (Fujifilm Wako), and BrdU (Santa Cruz Biotechnology) and appropriate isotype controls were used. Slides were imaged with Nikon A1 confocal microscope and analyzed through the ImageJ or NIS Elements (Nikon) software.
Nguyen A.V., Bagood M.D., Wang M., Caryotakis S.E., Smith G., Yee S., Shen H., Isseroff R.R, & Soulika A.M. (2022). Montelukast, an antagonist of cysteinyl leukotrienes signaling, impairs burn wound healing. Plastic and reconstructive surgery, 150(1), 92e-104e.
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4

Multimarker Immunofluorescence Tissue Staining

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Slides underwent de-paraffinization and were rehydrated, then blocked with 5% BSA, incubated with primary antibodies (FOXC1, 1:100, Onconostic Technologies; PRL-R, 1:100, sc-30225, Santa Cruz; Keratin 5, 1:100, 905501, Biolegend; Keratin 8, 1:100, DSHB) overnight at 4 °C and secondary antibodies (Alexa Fluor 488 and 594; 488 and 546, Life Technologies) for 1 hour at room temperature. The nuclei were stained with DAPI (Life Technologies). Images were acquired with a fluorescence microscope (ThermoFisher EVOS FL Auto Cell Imaging System).
Gao B., Qu Y., Han B., Nagaoka Y., Katsumata M., Deng N., Bose S., Jin L., Giuliano A.E, & Cui X. (2017). Inhibition of lobuloalveolar development by FOXC1 overexpression in the mouse mammary gland. Scientific Reports, 7, 14017.
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5

Histological Analysis of Mouse Ear Tissue

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Serial sections from mouse ears embedded in formalin‐fixed paraffin were processed for H&E staining and for immunofluorescence. Paraffin sections were processed for their deparaffinization and antigen retrieval. Primary antibodies specific for Ki67 (Master Diagnostica, clone SP6, dilution 1:5), Keratin 5 (Biolegend, ref # 905901, dilution 1:1000), Involucrin (Covance, ref # PRB‐14℃, dilution 1:1000), S100A9 (Santa Cruz, ref # SC8115, dilution 1:100), Ly6b (BioRad, ref # MCA771GA, dilution 1:200) and S. aureus (Abcam, ref #ab20920, dilution 1:200) were incubated overnight (O/N) at 4ºC. Sections were then washed three times with PBS and incubated with secondary antibodies conjugated to the appropriate fluorophores (Life Technologies) for 1 h at RT. Sections were then washed three times with PBS and mounted with gel mount to be analysed by inverted fluorescence microscope (Nikon) or confocal microscope (TCS‐SP5, Leica Microsystems).
Gago‐López N., Lagunas Arnal C., Perez J.J, & Wagner E.F. (2021). Topical application of an amygdalin analogue reduces inflammation and keratinocyte proliferation in a psoriasis mouse model. Experimental Dermatology, 30(11), 1662-1674.
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6

Immunohistochemical Analysis of Skin Samples

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Paraffin-embedded skin biosies were cut at 5 μm on frosted microscope slides. Slides were deparaffinized and then rehydrated. Skin sections were then blocked with 5% BSA in Super Block (ScyTek Laboratories, Logan, UT). Slides were then stained with antibodies directed against Loricrin (Abcam, Cambridge, MA; cat #Ab85679, 1:500); Filaggrin (Biolegend, Dedham, MA; cat #905801, 1:500); Keratin 5 (Biolegend, cat #90550, 1:2000); IL-1b (Biotechne, Minneapolis MN; cat #AF-401, 1:500); IL-1a (Biotechne, cat #AF-400, 1:1000), IL-36a (Biotechne, cat #AF-2297, 1:1000); Ki67 (EMD Millipore Corporation, Temecula CA; cat #AB9260 1:200), Myeloperoxidase (ThermoFisher, Rockford, IL; cat #PA5–16672, 1:200) or equal amounts of isotype control antibodies. Slides were incubated at 4°C overnight and then washed with PBS /Tween 0.05%. Secondary Cy-3 conjugated antibody (Jackson Labs; West Grove, PA) was added for 1 hour and cell nuclei were visualized with DAPI (Sigma). Images were taken with a Leica Microscope (Wetzlar, Germany) at 40x magnification using SlideBook software (Intelligent Imaging Innovations, Denver, CO). Mean fluorescence intensity (MFI) was determined for each exposure group using SlideBook software and was reported as mean MFI ± SE.
Brauweiler A.M., Goleva E, & Leung D.Y. (2019). Staphylococcus aureus Lipoteichoic Acid Damages the Skin Barrier through an IL-1 mediated Pathway. The Journal of investigative dermatology, 139(8), 1753-1761.e4.
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7

Immunostaining Protocol for Mouse Epidermis

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Immunostaining experiments were carried out on 7 micron frozen sections of mice epidermis. Air-dried frozen sections were post fixed in 4% paraformalydehyde. Sections were incubated with following primary antibodies: Keratin 5 (905501, Rabbit polyclonal, Biolegend), E-cadherin (180223, Mouse monoclonal IgG1kappa, Life technologies), Claudin 1 (37-4900, Mouse IgG1, Thermofisher Scientific), Ki67 (ab15580, Rabbit polyclonal, Abcam). Primary antibodies were used at 1:200 dilution in 4% NGS block overnight. Sections were incubated with the following secondaries: Alexafluor 488 anti-rabbit (A-11008, Thermofisher Scientific), Alexafluor 488 (A-11029, Thermofisher Scientific), Alexafluor 594 anti-mouse (A-11032, Thermofhisher scientific) and nuclear stain DAPI at 1:200. Confocal imaging was performed using the Nikon TI-E Inverted microscope at the Cincinnati Children’s Confocal Imaging Core. Images were analyzed using the Nikon Imaging Software Elements (Version 4.4) and ImageJ (Version 1.52) was used to quantify immunostaining images as appropriate.
Stevens M.L., Zhang Z., Johansson E., Ray S., Jagpal A., Ruff B.P., Kothari A., He H., Martin L.J., Ji H., Wikenheiser-Brokamp K., Weirauch M.T., Supp D.M., Biagini Myers J.M, & Khurana Hershey G.K. (2020). Disease-associated KIF3A variants alter gene methylation and expression impacting skin barrier and atopic dermatitis risk. Nature Communications, 11, 4092.
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