After the indicated stimulation, cells were fixed with cold methyl alcohol for 5 min at −20 °C. Cells were then permeabilized in phosphate buffer solution (PBS, pH7.4) containing 5% goat serum and 0.1% Triton X-100 for 60 min at room temperature. Next, the monoclonal antibodies anti-keratin-18, anti-keratin-8, anti-keratin-8-Ser431, anti-keratin-8-Ser73, anti-keratin-18-Ser52 (Abcam, Cambridge, UK), or anti-pan-keratin (Cell Signaling Technology, USA) were added overnight at 4 °C. Cells were then incubated with FITC (fluorescein isothiocyanate isomer)-conjugated or Alexa Flour 594-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h without light. After washing with PBS, imaging was performed using a fluorescence microscope (Leica Microsystems, Wetzlar, Germany) equipped with a 100× objective.
Anti pan keratin
Manufactured by Cell Signaling Technology
Sourced in United Kingdom
Anti-pan-keratin is a laboratory reagent used for the detection and localization of keratin proteins in cells and tissues. It is a broad-spectrum antibody that recognizes multiple keratin subtypes, making it a useful tool for identifying epithelial cells in various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscope, by Leica (1 mentions)
Anti keratin 8, by Abcam (1 mentions)
Paraformaldehyde (pfa), by Wuhan Servicebio Technology (1 mentions)
Anti vimentin, by Cell Signaling Technology (1 mentions)
Anti cd31, by Cell Signaling Technology (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Cleaved caspase 3 asp175, by Cell Signaling Technology (1 mentions)
Orca r2, by Hamamatsu Photonics (1 mentions)
Aperio at2 scanner, by Leica (1 mentions)
Lsm 700 meta, by Zeiss (1 mentions)
Anti kim 1, by Abcam (1 mentions)
Anti ngal, by Abcam (1 mentions)
5 protocols using anti pan keratin
1
Immunofluorescence Staining of Keratins
2
Quantifying Cellular Composition in Tumor Microenvironment
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To determine the proportion of epithelial cells, endothelial cells and fibroblasts in the CC TME, one SCC sample and one ADC sample were fixed in 4% paraformaldehyde (Servicebio) for paraffin embedding. Thereafter, 4‐µm‐thick sections were prepared. The following antibodies and dilutions were used to detect proteins: anti‐pan‐keratin (mouse, 1:250, 4545, Cell Signaling Technology), anti‐vimentin (rabbit, 1:100, 5741, Cell Signaling Technology), and anti‐CD31 (rabbit, 1:200, 77699, Cell Signaling Technology). According to the percentage of positive cells, the IHC staining area score was divided as follows: 0 point (<5%), 1 point (5–25%), 2 points (26–50%), 3 points (51–75%) %), and 4 points (76–100%), whereas the IHC staining intensity was divided as follows: 0 point (negative); 1 point (weak positive); 2 points (positive); 3 points (strong positive). Immunoreactive scores were calculated by multiplying the area and intensity scores.
3
LUAD Tissue Microarray Staining and Imaging
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2 mm diameter circular tissue columns were extracted from the paraffin blocks of the 55 LUAD tissues and embedded into single blocks to produce tissue microarrays (TMAs). Each TMA contains one tonsil tissue and 11 tissues from one of the LUAD subtypes. Sections from TMAs were stained with primary, and fluorescent labeled secondary antibodies. We used the following dye and antibodies: Hoechst 33342 (Thermo Fisher, #H1399), anti-Pan-Keratin (Cell Signaling, #4545), cleaved-Caspase-3 (Asp175) (Cell Signaling, #9661), and EpCAM (MOC-31) (Invitrogen, #MA5-12442). Fluorescent images were obtained through an upright microscope (BX63; Olympus) with a cooled charge-coupled device (CCD) camera (ORCA-R2; Hamamatsu Photonics) and a 10x, 0.40 NA, objective (Olympus). Obtained images were analyzed with Spectral Unmixing Plugin installed in ImageJ.35 (link)
4
IHC Assay for Pan-Keratin and FOSL1
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IHC assay was performed with Anti‐Rabbit and Anti‐Mouse HRP/DAB (ABC) IHC DETECTION Kit (Abcam, ab64261 and ab64259) according to the manufacturer's instructions. The primary antibodies used in IHC were anti‐Pan‐Keratin (Cell Signaling, 4545S), anti‐FOSL1 (Abcam, ab232745). Images of specimen sections were obtained with the Aperio AT2 Scanner (Leica Biosystems, Germany), and the data were analyzed by imageJ software. The IHC score was calculated by the formula as the addition of product of different density and percentage of cell staining.
5
3D Kidney Organoid Protein Expression
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Expression levels of Tamm-Horsfall protein (THP), pan-keratin (Ker), NGAL, and KIM-1 were measured using immunofluorescence. Briefly, after a 2-week differentiation period, 3D kidney organoids (3D-embedded) in PBS were pipetted very gently to separate them from the Matrigel matrix for whole organoid stain. Whole organoids seeded (37°C, 16 h) onto an 8-chamber cell-culture slide were fixed (10 min) in 4% paraformaldehyde solution and washed in PBS. Cells were permeated (5 min) in 0.5% Triton X-100/PBS, blocked (20 min) with 5% BSA/PBS, and incubated (4°C, 16 h) in diluted primary antibodies: FITC-conjugated anti-THP (Cedarlane Laboratories, Burlington, NC, USA), anti-NGAL (Abcam, Cambridge, UK), anti-KIM-1 (Abcam) and anti-pan-keratin (Cell Signaling Technology, Danvers, MA, USA). Finally, PBS-washed cells were exposed (25° C, 1 h) to secondary antibodies tagged with Alexa Fluor 488 or 594 (1:200; Invitrogen [Thermo Fisher], Waltham, MA, USA). Images were captured using a confocal microscope (LSM 700 META; Carl Zeiss AG, Oberkochen, Germany) and analyzed using proprietary software (LSM Image Browser).
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