VERO (ATCC®CRL-1586) and PK-15 cells (ATCC®CCL-33) were cultured in Dulbecco’s modified Eagle’s medium (CORNING, USA) supplemented with 10% fetal bovine serum (Gibco) at 37°C in a humidified atmosphere containing 5% CO2. The PRV strain ZJ 01 was isolated from a pig herd at China in 2012 (Gu et al., 2015 (link)), which was used for all experiments. The PRV were proliferated in PK-15 cells and stored at −80°C. β-Streptococcus equinus strain SheepZ001 were cultured in Tryptone soybean broth (TSB) + 0.05% fetal bovine serum (FBS) medium at 37°C, 220 rpm. Pasteurella multocida HB01 (Serotype A) were cultured in Brain-heart extract broth (BHI) medium at 37°C, 220 rpm. Bacillus subtilis 168, WB800 and other Bacillus subtilis group isolated from sheep nasal cavity were cultured in Luria-Bertani (LB) medium at 37°C, 220 rpm. Anti-PRV gB-protein monoclonal antibodies (1B1, prepared and stored in our laboratory), anti-GAPDH antibody (Proteintech), anti-cytokeratin18 (Abcam), anti-claudin 1(Abcam), anti-caspase3 (Proteintech); anti-caspase9 (Proteintech); anti-ISG15 antibody (Abcam), anti-PCNA antibody (Abcam), anti-CD3 antibody (Dako), anti-IgA antibody (Abcam) and anti-CD208 antibody (Dendritics) was used in the study.
Anti cytokeratin 18
Manufactured by Abcam
Sourced in United States
Anti-cytokeratin 18 is a lab equipment product used for the detection and identification of cytokeratin 18 in biological samples. Cytokeratin 18 is a type of intermediate filament protein found in the cytoskeleton of epithelial cells.
Automatically generated - may contain errors
Lab products found in correlation
Live dead fixable blue dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Anti uea 1, by Vector Laboratories (2 mentions)
Live dead staining kit, by Thermo Fisher Scientific (2 mentions)
Anti cd24, by Thermo Fisher Scientific (2 mentions)
Tri reagent, by Thermo Fisher Scientific (2 mentions)
Anti cd45, by BioLegend (2 mentions)
Aria 2 cell sorter, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Anti claudin 1, by Abcam (1 mentions)
Anti pcna antibody, by Abcam (1 mentions)
Anti caspase 3, by Proteintech (1 mentions)
Anti caspase 9, by Proteintech (1 mentions)
Anti cd3 antibody, by Agilent Technologies (1 mentions)
Anti iga antibody, by Abcam (1 mentions)
Anti gapdh antibody, by Proteintech (1 mentions)
Dulbecco s modified eagle s medium, by Corning (1 mentions)
Anti pan cytokeratin, by Abcam (1 mentions)
Anti cytokeratin 13, by Abcam (1 mentions)
Anti cytokeratin 14, by Abcam (1 mentions)
6 protocols using anti cytokeratin 18
1
Porcine Reproductive Virus Research Protocol
2
Immunofluorescence Analysis of PCV2b Capsid
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Cells cultured in 60-mm dishes were fixed with stationary liquid (methanol and acetone, v/v = 1:1) for 20 min at −20°C. The cells were treated with 1% Triton X-100 in PBS for 10 min at room temperature. After blocking with 5% skimmed milk, cells were incubated with the primary antibodies at 4°C overnight. The following primary antibodies were used at a dilution of 1:500: anti-pan-cytokeratin, anti-cytokeratin 13, anti-cytokeratin 14, anti-vimentin, anti-cytokeratin 18 (All obtained from Abcam, Cambridge, MA, USA), and anti-PCV2b Cap antibody (kindly provided by Dr. Shuanghui Yin, Lanzhou Veterinary Research Institute, China). Protein staining was detected using secondary fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG or FITC-conjugated goat anti-rabbit IgG (1:100, Tianjin Sungene Biotech, China) for 1 h. The cells were observed and imaged by Evos f1 fluorescence microscope (Advanced Microscopy Group, Mill Creek, WA).
3
Isolation and Sorting of Intestinal Epithelial Cells
4
Evaluating Epithelial-Mesenchymal Transition
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Where indicated, the following drugs were used: MG132 (10 μM, Sigma, Saint Louis, MO, USA), cycloheximide (20 nM, Sigma, Saint Louis), CsA (2 μg/ml or 20 μg/ml, Sangon, Shanghai, China), and Leptomycin B (5 ng/ml, Beyotime, Shanghai, China). Rabbit anti-Flag, anti-GFP, anti-HA, and mouse anti-Flag antibodies were purchased from Sigma (Sigma, Saint Louis, MO, USA). Mouse anti-E-cadherin, anti-Cytokeratin 18, and rabbit anti-F-actin and anti-Vimentin antibody, rabbit anti-N-cadherin, and anti-SNAI1 antibodies were obtained from Abcam (Abcam, Cambridge, MA, USA). Rabbit anti-PPIL2 antibodies were purchased from Thermo Fisher Scientific (Thermo Fisher, Massachusetts, USA). Anti-fibronectin antibody was purchased from Wanleibio (Wanleibio, Shenyang, China).
5
Isolation and Sorting of Intestinal Epithelial Cells
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The epithelial fraction of the small intestine was isolated as described previously9 (link). Briefly, mice were euthanized, and small intestine was collected in cold PBS. Tissues were washed with cold PBS twice, chopped and transferred to new tubes followed by incubation in stripping buffer (10% FBS, 15 mM HEPES, 5 mM EDTA, in 1X HBSS) for 20 min at 37°C. The tissues were vortexed for 15 sec and passed sequentially through 100 microns and 40 microns filters. The collected cells were washed with cold PBS and stained with live/dead staining kit (live/dead fixable blue dead cell stain kit, Life Technologies) according to manufacturer’s protocol. After live/dead staining, cells were stained with anti-CD24 (eBioscience; clone 30-F1), anti-CD45 (Biolegend, clone ), anti-UEA-1 (Vector labs), anti-cytokeratin 18 (Abcam; clone C-04). Goblet cells were sorted using BD ARIA II cell sorter (BD Biosciences) by gating on CD45−CD24−CK-18+UEA-1+, while the remaining IECs were collected by gating on CD45−CD24−CK-18−UEA-1− and collected in RPMI containing 10% FBS21 (link). The collected cells were centrifuged and resuspended in Tri Reagent (Invitrogen) for RNA extraction. Data were processed using FACSDiva software (BD) and FlowJo (FlowJo).
6
Immunocytochemistry Staining Protocol
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Cells were fixed in 4% paraformaldehyde for 20 min, permeabilized in 0.1% Triton X-100 (Sigma-Aldrich) for 20 min, and blocked in 3% bovine serum albumin (BSA; Sigma-Aldrich) for 1 h. The cells were then incubated with the primary antibody at 4°C overnight. The mouse anti-a-fetoprotein (Cell Signaling Technology, Beverly, MA, USA), rabbit anti-albumin (Abcam, Cambridge, UK), rabbit anti-cytokeratin 7 (Abcam), anti-cytokeratin 18 (Abcam), and sheep anti-FIX (Molecular Innovations, Novi, MI, USA) primary antibodies were used at a dilution of 1:100, and the rabbit anti-cytokeratin 19 (Abcam) primary antibody was used at a dilution of 1:1,000. After washing with PBS-0.02% Tween-20 (Sigma-Aldrich), cells were incubated with FITC-or PE-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) diluted to 1:200 for 2 h and then stained with 4¢,6-diamidino-2phenylindole (DAPI; Biogenex, San Ramon, CA) to stain the cell nuclei.
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