Cells were seeded in a 96 well plate at 10,000/well in growth media. After 24 hours, cells were fixed in 10% neutral formalin buffer (NBF) for an additional 24 hours, permeabilized with methanol for 10 minutes at −20°C. For primary incubation with mouse monoclonal antibodies, cells were blocked in PBS containing 3.4% mouse FAB fragment for (Jackson Laboratory cat no. 715 001 003) for 1 hour. For all other antibodies, cells were blocked in PBS containing 3% FBS. Cells were incubated with primary antibodies at a 1: 3 dilution to Fibroblast Specific Protein 1 (Fsp1) (Abcam cat no. ab75550), or at a 1:100 dilution to: pan-cytokeratin (Santa Cruz Biotechnology cat no. sc 8018), α−smooth muscle actin (α-sma) (Abcam cat no. 7187), N-cadherin (Santa Cruz Biotechnology cat no. 7339) or Von Willebrand Factor 8 (VWF8, Millipore cat no. Ab7356). pan-cytokeratin and α-sma were detected with secondary anti-mouse-hrp at 1:500 dilution for 2 hours in PBS/3% FBS and incubation with 3,3’-diaminobenzidine substrate (DAB, Vector Laboratories cat no. SK-4100). FSP1, N-cadherin and VWF8 were detected with secondary anti-rabbit-hrp at a 1:500 dilution and DAB substrate. Cells were counterstained with Mayer’s hematoxylin for 2 minutes. Images were captured using the EVOS FL Auto Imaging system.
Pan cytokeratin
Manufactured by Vector Laboratories
Pan-cytokeratin is a broad-spectrum antibody that recognizes multiple cytokeratin subtypes. Cytokeratins are intermediate filament proteins expressed in epithelial cells. Pan-cytokeratin can be used to identify and label a wide range of epithelial cell types.
Automatically generated - may contain errors
Lab products found in correlation
α sma, by Vector Laboratories (2 mentions)
N cadherin, by Santa Cruz Biotechnology (1 mentions)
Pan cytokeratin, by Santa Cruz Biotechnology (1 mentions)
α smooth muscle actin α sma, by Abcam (1 mentions)
Normal goat or normal chicken serum, by Jackson ImmunoResearch (1 mentions)
Goat serum, by Vector Laboratories (1 mentions)
Lsm 700 confocal microscope, by Zeiss (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
Anti vegfr2, by Santa Cruz Biotechnology (1 mentions)
α sma, by Abcam (1 mentions)
3 3 diaminobenzidine substrate, by Vector Laboratories (1 mentions)
Anti pdgfrα, by Cell Signaling Technology (1 mentions)
Anti pan cytokeratin, by BioLegend (1 mentions)
Anti fsp 1, by Abcam (1 mentions)
3 protocols using pan cytokeratin
1
Immunocytochemical Characterization of Cell Types
2
Immunofluorescence and Immunohistochemical Analysis
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Immunofluorescence and immunohistochemical analysis (IHC) were performed using monoclonal antibodies against CD31 and pan-cytokeratin (Sigma). For immunofluorescence, after blocking with 5% normal goat or normal chicken serum (Jackson Immunoresearch) in PBS/Tween (PBST) (0.3% Triton) for 1 h at room temperature (RT), the sections were treated with anti-CD31 primary antibodies overnight at 4 °C. After three rinses with PBST, the sections were incubated for 1 h at RT with FITC-conjugated goat anti-rabbit secondary antibodies (1:500). After three rinses with PBST, the samples were counterstained with 4’ ,6-diamidino-2-phenylindole (DAPI) (1:10,000, Roche) (5 min at RT). The samples were washed with PBST once and mounted with the ProLong Gold anti-fade reagent (Invitrogen) in the dark. Fluorescent signals were visualized and digital images were obtained using the Zeiss LSM-700 confocal microscope (Carl Zeiss). For IHC, after blocking with 5% goat serum in PBST for 1 h at room temperature, the sections were treated with the pan-cytokeratin antibody overnight at 4 °C, then the peroxidase conjugated streptavidin complex method was performed, followed by the 3, 3′ diaminobenzidine (DAB) procedure according to the manufacturers’ protocols (VECTASTAIN Elite ABC Kit, Vector Lab).
3
Characterization of Fibroblast Phenotypes
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5000 fibroblasts were seeded per well in a 96 well plate in DMEM/10% FBS overnight. Cells were fixed in 10% neutral formalin buffer for 24 hours, permeabilized in methanol for 10 minutes at -20°C and blocked in PBS containing 3% FBS for 1 hour. Cells were then incubated with the following antibodies (1:100) for 24 hours in blocking buffer: anti-PDGFR-α (Cell Signaling Technology, cat no.5241), anti-FSP1 (Abcam, cat no. ab75550), α-SMA (Abcam cat no. 7817), anti-VEGFR2 (Santa Cruz Biotechnology, cat no.sc-393163) and anti-Pan-Cytokeratin (Biolegend, cat no.628602). Cells were washed in PBS 3 times and incubated with the following secondary antibodies (1:1000): anti-rabbit-biotinylated (Jackson Laboratories, cat no.111-065) to detect PDGFR-α or anti-mouse-biotinylated (Vector Laboratories, cat no.BA-9200) to detect VEGFR2, α-SMA or Pan-Cytokeratin. Biotinylated antibodies were incubated with streptavidin bound to horseradish peroxidase (HRP) (Vector Laboratories, cat no. SK4100) for 30 minutes. FSP1 was detected using secondary anti-rabbit-HRP (1:500, Avantor, cat no. 10150-732). Protein expression was detected through HRP reaction to 3, 3 -diaminobenzidine substrate (Vector Laboratories, cat no.SK4100).
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