Tumor tissue or cultured cells were fixed in formalin and paraffin embedded according to routine protocol. Deparaffinization and rehydration of tissue sections of 4 μm thickness was performed according to standard procedures. Sections were subjected to epitope retrieval in a PT Link module (Dako, Santa Clara, CA, USA) and immunohistochemical stainings were performed using an Autostainer Plus (Dako) according to the manufacturer’s standard protocol. Antibodies used were: cytokeratin 7 (Dako, M7018, 1:100) cytokeratin 19 (Ventana, Basel, Switzerland, 760-4281, prediluted), vimentin (Ventana, 790-2917, prediluted), CD10 (Ventana, 790-4506, prediluted) CD31 (Ventana, 760-4378, prediluted), CD68 (Dako, M0814, 1:1000), CD163 (Ventana, 760-4437, prediluted), and CXCL16 (Abcam, Cambridge, UK, ab101404, 1:100). Sections were stained with hematoxylin and eosin or counterstained with hematoxylin.
Cytokeratin 7
Manufactured by Agilent Technologies
Sourced in Switzerland, Japan, United States, Austria
Cytokeratin 7 is a protein marker used in immunocytochemistry and immunohistochemistry applications. It is a member of the cytokeratin family of intermediate filament proteins, which are structural components of cells.
Automatically generated - may contain errors
Lab products found in correlation
Vimentin, by Agilent Technologies (2 mentions)
Cxcl16, by Abcam (1 mentions)
Vimentin, by Roche (1 mentions)
Autostainer plus, by Agilent Technologies (1 mentions)
Pt link module, by Agilent Technologies (1 mentions)
Cd163, by Roche (1 mentions)
Bcl 2, by Agilent Technologies (1 mentions)
Synaptophysin, by Agilent Technologies (1 mentions)
Ae1 ae3, by Agilent Technologies (1 mentions)
Dako omnis autostainer, by Agilent Technologies (1 mentions)
E cadherin, by Agilent Technologies (1 mentions)
Alexa 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Protein blocking solution, by Agilent Technologies (1 mentions)
Mouse anti cytokeratin 7, by Agilent Technologies (1 mentions)
Aquaporin 1, by Abcam (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Percoll, by Thermo Fisher Scientific (1 mentions)
Dispase dnase, by Thermo Fisher Scientific (1 mentions)
Anti cd90, by Agilent Technologies (1 mentions)
8 protocols using cytokeratin 7
1
Immunohistochemical Profiling of Formalin-Fixed Paraffin-Embedded Tissue Sections
2
Immunohistochemical Staining Validation
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Ten well-characterized antibodies [AE1/AE3, BCL2, CD20, CD3, CD68, cytokeratin 7, E-cadherin, Ki-67, MSH2, and synaptophysin (Dako, Carpinteria, CA)] were selected to represent a range of nuclear, cytoplasmic, and membrane staining patterns in a variety of tissues (Table 1 ). All antibodies were in the ready-to-use formulation, and all other Dako reagents [heat-induced epitope retrieval solutions, buffers, labeled polymer, 3,3′-diaminobenzidine (DAB) and hematoxylin counterstain] were designed for use with the Dako Omnis Autostainer. The same antibody solutions were used across all 12 staining runs at 2 locations, and all staining runs were performed within 21 days. All reagents for all staining runs were from single lots.
3
Placental Cytokeratin 7 and PTTG1 Profiling
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To analyze Cytokeratin 7 (Dako) and PTTG1 (Invitrogen) expression in placenta, placental tissues were embedded in OCT compound (Tokyo, Japan, Sakura Finetechnical Co. Ltd), and preserved at -80°C. Two serial sections of 6-μm thick were made of the same tissue specimen, and then fixed in 100% methanol. One section was stained with Hematoxylin and Eosin (H%E) solution. Another section was incubated in protein blocking solution (Dako) at room temperature for 30 min, and then incubated overnight at 4°C with mouse anti-cytokeratin7 (1:100 dilution; Dako) and rabbit anti-PTTG1 (1:100 dilution; Invitrogen), followed by 1 h of incubation at room temperature with Alexa 594-conjugated (1:200 dilution; Invitrogen) and Alexa 488–conjugated secondary antibody (1:200 dilution; Invitrogen). DAPI (Vector Laboratories, Burlingame, CA, USA) was used as a counterstain. Images were analyzed using an EVOS fl microscope (AMG, Mill Creek, WA, USA).
4
Determining Tetraspanin-8 Expression in Renal Tubules
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To identify the types of tubule cells that express tetraspanin-8, we performed immunohistochemistry on five renal tissues associated with IgAN. Adjacent sections were further stained for cytokeratin 7 (a marker of distal and collecting tubules, 1:200 dilution; Dako), aquaporin 2 (a marker of collecting tubules, 1:500 dilution; Lifespan, Seattle, Wash., USA) or aquaporin 1 (a marker of proximal tubules, 1:200 dilution; Abcam).
5
Isolation of Primary Human Trophoblasts
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Primary human first trimester trophoblasts were isolated as previously described [33 (link),41 (link)]. Briefly, first trimester placental tissue was digested with Dispase/DNAse and Trypsin (Gibco, Invitrogen, Carlsbad, CA, USA). After Percoll (Gibco) gradient centrifugation, fibroblasts and common leukocyte-antigen expressing cells were removed by negative immunoselection with anti-CD90 (Dako, Glostrup, Denmark) and anti-CD45 conjugated magnetic beads (Thermo Fisher Scientific, Rockford, IL, USA), respectively. Purity of the trophoblast preparations was determined by human chorionic gonadotropin secretion as well as positive cytokeratin 7 (Dako, 1:750) and negative vimentin (Dako, 1:250) immunostaining. Only isolations with a purity ≥95% were used.
6
Immunohistochemical Staining of Macaque Tissues
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Sectioned tissues were fixed in 3.7% formaldehyde in PIPES buffer and blocked with normal donkey serum prior to staining. For adherens junction identification in macaque tissues, HECD1 (a gift from the laboratory of Dr. Kathy Green at Northwestern University) was utilized. To identify target cells, rhesus macaque tissue was stained with MCD1 (Santa Cruz) CD4 (Cell Marque) and CD68 for macrophages (DakoCytomation). Additional antibodies revealed cytokeratin-7 (DakoCytomation) staining in simple columnar tissue. To confirm that all PA-GFP fluorescence was associated with viral proteins, p24 (AG3.0 National Institutes of Health AIDS Research and Reference Reagent Program; Jonathon Allan), and p17 (Capricorn) antibodies were used [56 (link)]. Secondary antibodies, Rhodamine RedX (Jackson ImmunoResearch) and Cy5 (Jackson ImmunoResearch), were also utilized. Antibody specificity was determined by negative results with respective isotype control antibodies. Hoechst DAPI (Invitrogen) was used for DNA staining and wheat germ agglutinin (Invitrogen) highlighted cellular glycoproteins. After staining, mounting medium (DakoCytomation) and coverslips were applied and sealed with nail polish.
7
Isolation and Purification of Placental Trophoblasts
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Trophoblasts were isolated as described by Chen et al. 22 In brief, after mincing placental villous tissue from healthy controls (n = 6) and women with GDM (n = 6), the tissue was digested with a trypsin/dispase/DNase solution (Gibo, Roche, Sigma) for 90 min. The cell suspension was centrifuged on a Percoll gradient (Sigma, St Louis, USA) at 4 °C for 30 min at 300 g without brakes. Trophoblast-enriched layers were purified by immunodepletion of contaminating cells using beads conjugated to MCA-81 antibody against HLA-A, B and C (Serotec, Puchheim, Germany). Trophoblasts (3 × 10 6 ) were seeded in each well of a six-well dish in 2 ml DMEM containing 10% FCS and 1% penicillin/streptomycin (Gibco, Lofer, Austria) at 37 °C and 8% oxygen. After 48 h, viability was tested by measuring secreted β-hCG levels in the culture supernatant (Siemens). Purity was determined by immunocytochemical staining for the trophoblast marker cytokeratin 7 (Dako, Vienna, Austria) and for the mesenchymal cell marker vimentin (Dako). 23 Only preparations with a purity ≥ 99% were used.
8
Immunocytochemical Analysis of Cell Proteins
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For immunocytochemistry studies, cells were seeded in 24-well plate on glass-coverslips, at a cellular density of 1.5 × 10 4 cells/well and fixed with cold methanol after treatment with differentiation stimulus for 6 d. The expression of proteins was analysed using an avidin-biotin alkaline phosphatase complex technique (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA). To prevent non-specific binding, slides were incubated in blocking serum (normal serum) and slides were incubated with primary antibodies vimentin (1:500, sc 6260; Santa Cruz Biotechnology) and cytokeratin-7 (1:100, M 7018; Dako) overnight at 4°C. It was followed by incubation with biotinylated secondary antibody and with Vectastain ABC-AP reagent, according to the manufacturer's instructions. The reaction was developed Sigma Fast RedTM tablets (Sigma-Aldrich). Mayer's haematoxylin solution (Sigma-Aldrich) was used as counterstaining and slides were mounted in Aquamount medium (BDH Laboratory Supplies, Pl, UK). Negative controls were performed by the replacement of the primary antibodies by rabbit IgG. The results are the mean of three independent experiments.
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