Keratin 14

Manufactured by Cell Signaling Technology

Sourced in United States

Keratin 14 is a protein marker that is expressed in basal cells of stratified epithelia. It is commonly used in immunohistochemistry and immunocytochemistry applications to identify and characterize these cell types.

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3 protocols using keratin 14

Epidermal Protein Expression Analysis

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Facial hair was removed using Nair® hair removal cream and skin around the cheek and eyelid area was dissected out in one piece, snap frozen and stored at −80°C until usage. The dissected skin samples were then carefully scraped with a curette to selectively remove only the epidermal layer, which was lysed in RIPA buffer containing protease inhibitors. Equal amounts of the lysates were separated on SDS-PAGE and the blots stained with primary antibodies were visualized using IRDye linked secondary antibody in Odyssey SA scanner (LICOR Biosciences, Lincoln, NE) as previously described (Qu et al., 2011 (link)). Antibodies used are specific to Actin (Abcam, Cambridge, MA), GFP (a gift from Dr. Pamela Silver, Harvard Medical School, Boston, MA), Ki67 (BD Pharmingen San Diego, CA), Keratin 10, Keratin 14, phospho-4EBP1 ^T37/46, phospho-S6^S235/236, phospho-Akt^S473, Pten (all from Cell Signaling Technology, Beverly, MA), phospho-Histone H3 (Upstate, Temecula, CA), Fgf1, Fgf2, Fgf7, and Fgf10 (all from Santa Cruz Biotechnology, Santa Cruz, CA). At least three mice of each genotype were analyzed.

Hertzler-Schaefer K., Mathew G., Somani A.K., Tholpady S., Kadakia M.P., Chen Y., Spandau D.F, & Zhang X. (2014). Pten loss induces autocrine FGF signaling to promote skin tumorigenesis. Cell reports, 6(5), 818-826.

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Western Blot Analysis of Protein Markers

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Protein extracts were prepared as previously described^{22 (link)}. Overall, 10 μg of samples were electrophoresed and blotted to PVDF membrane (Bio-Rad, Hercules, CA, USA). Western blot was performed using primary antibodies against 15-lipoxygenase-1 (Mouse, Abcam, Cambridge, UK), PLIN1 (Rabbit, Cell Signaling, Danvers, MA, USA), Keratin 14 (Rabbit, Cell Signaling), phospho-RIP3(Rabbit, Abcam), RIP3(Rabbit, Cell signaling), ZBP1 (Rabbit, Cell Signaling), cleaved Caspase-3 (Asp175) (Rabbit, Cell Signaling), Caspase 3(Rabbit, Cell Signaling), Caspase 7(Rabbit, Cell Signaling), Tubulin (Cell Signaling), and β-actin (Mouse, Santa Cruz Biotechnology, Dallas, TX, USA) and secondary anti-mouse and anti-rabbit horse-radish peroxidase antibodies (Cell Signaling) as described previously. The blots were visualized with SuperSignal West Dura Substrate (ThermoFisher Scientific).

Kim S.N., Akindehin S., Kwon H.J., Son Y.H., Saha A., Jung Y.S., Seong J.K., Lim K.M., Sung J.H., Maddipati K.R, & Lee Y.H. (2018). Anti-inflammatory role of 15-lipoxygenase contributes to the maintenance of skin integrity in mice. Scientific Reports, 8, 8856.

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Histological Analysis of Mouse Skin

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Dorsal skin of mice was processed for histological sections, and 5-μm-thick paraffin sections were subjected to H/E staining or immunohistochemical analysis. The antibodies used for immunochemical detection were anti-15-lipoxygenase-1 antibody (Abcam), Perilipin A (Cell Signaling), Keratin 14 (Cell Signaling), Keratin 15 (Cell Signaling), PDGFRA (R&D systems), smooth muscle actin-FICT (Sigma), HMGB1 (Cell Signaling), tdTomato (Clonetech), Ly6C (Biolegend) and F4/80 antibody (AbD Serotec). The secondary antibodies used were donkey anti-mouse-Alexa Fluor 488, donkey anti-rabbit-Alexa Fluor 594/Alexa Fluor 488, donkey anti-goat-Alexa Fluor 594, and donkey anti-rat-Alexa Fluor 594 (ThermoFisher Scientific, Molecular Probes). The omission of primary antibody or normal rabbit, rat, goat, or mouse IgG controls (Santa Cruz Biotechnology) was used as a negative control. DAPI (Sigma) was used for nuclear counterstaining. Cells were imaged on a Zeiss confocal laser-scanning microscope (LSM 710 META, Zeiss, Jena, Germany).

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