The 4% formaldehyde-fixed and dehydrated skin tissue samples were embedded in paraffin wax and cut into 5-µm-thick sections. The skin sections were incubated with a primary antibody against human KRT10 (1:100; Abcam, Cambridge, UK), followed by incubation with the adequate secondary antibody. After staining, sections were counterstained with hematoxylin to provide contrast. For immunofluorescence staining, the skin tissues were quench-frozen and embedded in OCT. The cryosections (10 µm) were fixed in chilled acetone for 15 min and incubated overnight at 4 °C with anti-S100A9 (1:400; Abcam, Cambridge, UK), anti-HBD-2 (1:400; Abcam, Cambridge, UK), anti-Filaggrin (1:400; Abcam, Cambridge, UK), anti-Involucrin (1:400; Abcam, Cambridge, UK), anti-AO-1 (1:400; Abcam, Cambridge, UK), anti-CLDN-1 (1:400; Abcam, Cambridge, UK), anti-KRT6 (1:400; Abcam, Cambridge, UK), anti-KRT10 (1:400; Abcam, Cambridge, UK), and anti-KRT17 (1:400; Abcam, Cambridge, UK) primary antibodies, followed by Alexa Fluor 488- or 568-conjugated secondary antibodies and DAPI. The immunostained images were obtained using a digital camera (DP74; Olympus, Tokyo, Japan) coupled with an optical microscope (BX53; Olympus, Tokyo, Japan).
Anti krt17
Manufactured by Abcam
Sourced in United Kingdom, Germany
Anti-KRT17 is a primary antibody that binds to keratin 17 (KRT17), a type I intermediate filament protein. KRT17 is involved in the structural integrity of epithelial cells. This antibody can be used to detect and localize KRT17 in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti ythdf2, by Abcam (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Anti cldn 1, by Abcam (1 mentions)
Anti involucrin, by Abcam (1 mentions)
Anti hbd 2, by Abcam (1 mentions)
Anti krt10, by Abcam (1 mentions)
Anti s100a9, by Abcam (1 mentions)
Anti filaggrin, by Abcam (1 mentions)
Pierce protein a g magnetic beads, by Thermo Fisher Scientific (1 mentions)
Trypsin digestion, by Promega (1 mentions)
Q exactive, by Thermo Fisher Scientific (1 mentions)
Q exactive hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Reversed phase analytical column, by Thermo Fisher Scientific (1 mentions)
Mascot software, by Matrix Science (1 mentions)
Triton x 100, by Wuhan Servicebio Technology (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Wuhan Servicebio Technology (1 mentions)
Tcs sp8, by Leica camera (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
5 protocols using anti krt17
1
Immunohistochemical Analysis of Skin Markers
2
Immunoprecipitation of KRT17 and YTHDF2
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The cells transfected with the relevant plasmids were lysed in Pierce IP lysis buffer (Thermo Fisher Scientific) in the presence of a protease inhibitor cocktail (including protease inhibitors and phosphatase inhibitors; Thermo Fisher Scientific) for 30 minutes at 4°C. Centrifugation was conducted on cell lysates at 13,000 x g for 20 minutes at 4°C. The antibodies used in IP assays included anti-KRT17 (1:30, Abcam) and anti-YTHDF2 (1:50, Abcam). Next, the antibodies were applied to the 200-μL Pierce Protein A/G magnetic beads (Thermo Fisher Scientific) for 4 hours at 4°C to crosslink the antibodies. The antibody-crosslinked beads were mixed with the 500-μL cell lysates overnight at 4°C. After being washed with washing buffer (Cayman) five times, the beads were incubated with 2× sample loading buffer (Beyotime) and then boiled for 10 minutes. The lysates were resolved on SDS-PAGE (described below).
3
Proteomic Analysis of Anti-KRT17 IP
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The gel from the IP assay using anti-KRT17 (Abcam) was cut into tiny pieces. Peptides were dispersed in 0.1% formic acid (FA; Thermo Fisher Scientific) and 2% acetonitrile (ACN; Thermo Fisher Scientific) following trypsin digestion (Promega) and then were immediately placed onto a reversed-phase analytical column (Thermo Fisher Scientific). The gradient consists of increasing solvent B (0.1% FA in 80% ACN) from 5% to 50% in 40 minutes, while maintaining a consistent flow rate of 300 nL/min, as recommended by the manufacturer. Using Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific), MS analysis was conducted. Detection model included reflection mode and positive ion spectroscopy, the ion source accelerated voltage was 20 KV, and each peptide map was accumulated by 200 bombardments. Thermo Fisher Scientific's Q Exactive was used to perform tandem mass spectrometry (MS/MS) on the peptides, which was then connected online to perform ultra-performance liquid chromatography. The m/z scan range for MS scans was 350 to 1800 m/z. A fixed initial mass of 100 m/z was chosen. By using Uniprot Aedis Aegypti as a search engine, MASCOT software (Matrix Science) was used to identify proteins.
4
Immunofluorescence Analysis of KRT17 and YTHDF2
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Mouse MC38 and CT26 cell lines were chosen to evaluate cellular localization of KRT17 and YTHDF2 proteins. A cell slide (Thermo Fisher Scientific) was spread into a well of 12-well plate (Corning), and each well was inoculated with 2×104 MC38 or CT26 cells. 4% paraformaldehyde (Servicebio) was used to fix cells for 30 minutes at room temperature. 0.5% Triton X-100 (Servicebio) was added to break cell membrane for 30 minutes at room temperature and 1% BSA (Thermo Fisher Scientific) was used to block for 1 hour at room temperature. Specific primary antibodies, including anti-KRT17 (1:200, Abcam) and anti-YTHDF2 (1:200, Abcam) were incubated with the cells overnight at 4°C. Samples were added with Alexa 488- or 594-conjugated goat antibodies (1:1,000, Thermo Fisher Scientific) against rabbit or mouse IgG. The cells assessed using a confocal laser scanning microscope (Leica TCS-SP8) after being counterstained with DAPI (Merck Millipore).
5
Immunohistochemistry for Skin Biomarker Analysis
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The following antibodies and dilutions were used for immunohistochemical stainings: anti KRT17 (abcam, Berlin, Germany, 1:200), anti-BD 2 antibody (Peprotech, Rocky Hill, CT, USA, 1:100), anti-psoriasin antibody (abcam, Cambridge, MA, USA, 1:100), anti-GLUT1 antibody (abcam, Cambridge, MA, USA, 1:200), anti-NF-κB p65 antibody (F-6) (Santa Cruz, Heidelberg, Gemany) and the phospho-STAT3 (Tyr705; Cell Signaling Technologies, Leiden, the Netherlands). The secondary antibody multi-link-biotin (Agilent-Dako, Hamburg, Germany, 1:200), the streptavidin-HRP-label (abcam, Cambridge, MA, USA) and the AEC-substrate (Zytomed, Berlin, Germany) were used according to the manufacturer’s protocol. The fluorescence secondary antibodies Alexa Fluor 555 goat anti-mouse IgG and Alexa Fluor 555 donkey anti-rabbit IgG were from Thermo Fisher Scientific (Dreieich, Gemany). IL-22, IL-17A and TNF-α were from Peprotech (Rocky Hill, CT, USA). Dithranol and DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride) were from Sigma-Aldrich GmbH (Taufkirchen, Germany).
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