Mouse tissue (frozen whole skin and muscle samples) and 3D-keratinocytes were homogenized and sonicated in CelLytic MT Mammalian Tissue Lysis/Extraction Reagent (Sigma), and then centrifuged at 16,000g for 10 min at 4°C. The supernatants were harvested, and equal amounts of protein were subjected to SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were then incubated with primary antibodies to KRT5 (Abcam, Cambridge, UK), KRT10 (Abcam), KRT14 (Santa Cruz Biotechnology, Dallas, TX), desmoglein 1 (DSG1; GeneTex, San Antonio, TX), DSG2 (Abcam), desmocollin 3 (DSC3; Santa Cruz Biotechnology), α-tubulin (Cell Signaling), Akt (Cell Signaling), phospho-Akt (Ser473) (Cell Signaling), p70S6K (Cell Signaling), and phospho-p70S6K (Thr389) (Cell Signaling). After washing the membranes with 0.1% Tween 20 in PBS, they were exposed to a horseradish peroxidase-linked anti-rabbit IgG antibody (Cell Signaling). Bands were visualized with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK) and their intensities were quantified using the program Multi Gauge (Fujifilm, Tokyo, Japan).
Krt10
Manufactured by Abcam
Sourced in United Kingdom
KRT10 is a protein-coding gene that encodes for a type I keratin protein. Keratins are a group of structural proteins found in the cytoskeleton of epithelial cells. The KRT10 protein is a major component of the cytoskeleton in differentiated suprabasal keratinocytes.
Automatically generated - may contain errors
Lab products found in correlation
Krt15, by Santa Cruz Biotechnology (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Cellytic mt mammalian tissue lysis extraction reagent, by Merck Group (1 mentions)
Anti rabbit igg horseradish peroxidase linked antibody, by Cell Signaling Technology (1 mentions)
Krt14, by Santa Cruz Biotechnology (1 mentions)
α tubulin, by Cell Signaling Technology (1 mentions)
Phospho p70s6k thr389, by Cell Signaling Technology (1 mentions)
P70s6k, by Cell Signaling Technology (1 mentions)
Phospho akt ser473, by Cell Signaling Technology (1 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Multi gauge, by Fujifilm (1 mentions)
Krt19, by Santa Cruz Biotechnology (1 mentions)
Carl axio observer, by Zeiss (1 mentions)
Krt15, by Abcam (1 mentions)
Krt14, by Abcam (1 mentions)
Lichenan, by Merck Group (1 mentions)
Dextran standard, by Merck Group (1 mentions)
Uvasol, by Merck Group (1 mentions)
Rabbit anti mouse igg hrp, by Jackson ImmunoResearch (1 mentions)
Agilent vnmrs 600, by Agilent Technologies (1 mentions)
4 protocols using krt10
1
Western Blot Analysis of Epidermal Proteins
2
Immunofluorescence Staining of Corneal Cells
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Cells were fixed with 4% PFA for 20 minutes and permeabilized with 0.1% Triton X-100 for 10 minutes. Subsequently, the cells were blocked with 5% BSA for 60 minutes and incubated with primary antibodies against PAX6 (Thermo Fisher Scientific), TP63, Ki67 (Boster), KRT19, KRT15, KRT12 or KRT3 (Santa Cruz Biotechnology, Inc.) at instructed dilutions overnight in a cold room. Cells were washed with PBS at least 3 times and 15 minutes each time before incubation with the isotype-matching secondary antibodies (all from Invitrogen). The cell nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI). Bright-field and fluorescent images were captured using the Carl Zeiss Axio Observer. To check the expression of CEC markers on the recellularized DC and in sections of mouse skin tissues as controls, we processed the samples the same way and incubated with antibodies against human PAX6, KRT1, KRT10, KRT12, KRT14, KRT15, and VINCULIN (Abcam, Cambridge, United Kingdom), and mouse MHC Class I (Abcam), followed by secondary antibodies against the isotype of the primary antibodies and counterstaining with DAPI. Bright field and fluorescent images were captured with Carl Zeiss Axio Observer. All primary and secondary antibodies above were used at the dilution of 1:200 and 1:1,000, respectively.
3
Carbohydrate Analysis of Lichenan
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If not stated otherwise, all chemicals were purchased from VWR (Darmstadt, Germany). Lichenan was obtained from Sigma-Aldrich (Deisenhofen, Germany).
The following antibodies were used: β-actin (AC-15), mouse monoclonal (1:4000, 1 h at RT) from Sigma-Aldrich; KRT1 (1:200,000 for 2 h at RT), KRT10 (1:10,000 at 4 °C over night) and IVL (1:500,000 at 4 °C at RT), and rabbit monoclonals from Abcam; Rabbit anti-Mouse IgG HRP conjugate from Jackson ImmunoResearch; and Rabbit anti-Mouse IgG HRP (1:10,000 for 45 min at RT) from Jackson Immuno Research.
NMR spectra were recorded on an Agilent VNMRS 600 (Agilent Technologies, CA, USA). 1H and 13C-NMR measurements were obtained at 80 °C at 600 MHz and 150 MHz, respectively. 1H-13C-HSQC-NMR spectra were recorded at 20 °C. Data analysis was achieved with MestRneNova software version 10.0.0-14381. Sample concentration: 5 mg/mL D2O (Uvasol®, 99.8%, Merck, Darmstadt, Germany) and DMSO-D6 was added for referencing (δ 3.330 ppm).
Gel permeation chromatography was performed on a low pressure Sepharose®6 stationary phase (GE Healthcare, Freiburg, Germany) using standard dextrans (Sigma, Deisenhofen, Germany) for calibration. Each fraction was tested on the carbohydrate content accordingly [28 (link)].
The following antibodies were used: β-actin (AC-15), mouse monoclonal (1:4000, 1 h at RT) from Sigma-Aldrich; KRT1 (1:200,000 for 2 h at RT), KRT10 (1:10,000 at 4 °C over night) and IVL (1:500,000 at 4 °C at RT), and rabbit monoclonals from Abcam; Rabbit anti-Mouse IgG HRP conjugate from Jackson ImmunoResearch; and Rabbit anti-Mouse IgG HRP (1:10,000 for 45 min at RT) from Jackson Immuno Research.
NMR spectra were recorded on an Agilent VNMRS 600 (Agilent Technologies, CA, USA). 1H and 13C-NMR measurements were obtained at 80 °C at 600 MHz and 150 MHz, respectively. 1H-13C-HSQC-NMR spectra were recorded at 20 °C. Data analysis was achieved with MestRneNova software version 10.0.0-14381. Sample concentration: 5 mg/mL D2O (Uvasol®, 99.8%, Merck, Darmstadt, Germany) and DMSO-D6 was added for referencing (δ 3.330 ppm).
Gel permeation chromatography was performed on a low pressure Sepharose®6 stationary phase (GE Healthcare, Freiburg, Germany) using standard dextrans (Sigma, Deisenhofen, Germany) for calibration. Each fraction was tested on the carbohydrate content accordingly [28 (link)].
4
Immunofluorescence Staining of Cryosections
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For IF staining of sections, cryosections were made from frozen tissues embedded in OCT compound (Tissue Tek). The paired tissues were prepared on the same slide to ensure the same staining conditions. Slides were fixed for 10 min in 4% paraformaldehyde and blocked for 1 h in blocking buffer (2.5% normal donkey serum+2.5% normal goat serum+1% BSA+0.3% Triton X-100). Sections were then incubated with primary antibodies at 4°C overnight and with fluorochrome-conjugated secondary antibodies at room temperature for 1 h. Slides were then washed in PBS and mounted with Fluoromount-G mounting media (Invitrogen). Images were taken by Zeiss LSM 880 upright confocal multiphoton microscope. The following antibodies dilutions were used: KRT14 (chicken, Biolegend, 1:500), KRT15(mouse, Santa Cruz, 1:500), KRT10 (rabbit, Abcam, 1:500), KI67 (rabbit, Abcam, 1:500), PCNA(mouse, Servicebio, 1:200), BCL-2 (mouse, Biolegend, 1:200), CD45 (mouse, Biolegend, 1:200), E-CAD (rat, eBioscience, 1:200), Phospho-mTOR (Ser2448) (rabbit, CST, 1:50), FOS (rabbit, Abcam, 1:200), P63 (rabbit, Abcam, 1:200), AREG (rabbit, Abcam, 1:200), SERPINB2 (mouse, Novusbio, 1:200), NECTIN1(rabbit, Thermofisher, 1:100), CD96 (mouse, Santa Cruz, 1:50).
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