Calretinin

Manufactured by Agilent Technologies

Sourced in United Kingdom, United States, Belgium

Calretinin is a calcium-binding protein primarily used as a biomarker in the identification and characterization of various cell types, including neurons and certain cancer cells. It serves as a tool for researchers and clinicians to study cellular differentiation, development, and disease processes.

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6 protocols using calretinin

Histological Analysis of Intestinal Tissues

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Mice were euthanized by inhalation of a 5% CO₂ / 95% O₂ followed by 100% CO₂ for 4 min, and transcardially perfused with PBS to remove blood. Human and mouse formalin-fixed paraffin-embedded 5-μm-thick small intestine tissue sections were routinely stained for Hematoxylin-Eosin and Phosphotungstic acid-hematoxylin and immunostained as described [18 (link)] against smooth muscle actin (SMA, Dako, 1:200), CD117/c-kit (Dako, 1:50), calretinin (Dako, 1:200), NeuN (Millipore, 1:100), CD3 (Dako, 1:250), CD8 (Dako, 1:50) and glial fibrillary acidic protein (GFAP, Dako, 1:300). Immunoreactivity was detected using 3,3'-Diaminobenzidin or Liquid Permanent Red as chromogen. Pictures were taken with a Leica DM3000 microscope. Muscle thickness was measured on transversally cut intestinal sections (N = 40 for mice, N ≥ 25 for humans). Quantification of muscle wall thickness and ganglion cell density was performed blind to the genotype with ImageJ.

Yadak R., Boot M.V., van Til N.P., Cazals-Hatem D., Finkenstedt A., Bogaerts E., de Coo I.F, & Bugiani M. (2018). Transplantation, gene therapy and intestinal pathology in MNGIE patients and mice. BMC Gastroenterology, 18, 149.

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Histological Analysis of Ovarian Cancer

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Tumors excised from the peritoneal cavity of 32 patients with epithelial ovarian cancer (stage III and IV) as well as fragments of tumor-free peritoneum from the same patients were included in the study. The age of patients ranged from 42 to 60 years. The tissues were fixed in 4% formalin and were then processed as described in^{11 (link)}. The cancer tissue was identified using standard H + E staining. Peritoneal mesothelium located near the cancer cells was identified based on positive stainings of the D2-40, Wt-1, HBME-1, and calretinin with specific antibodies (Dako, Santa Clara, CA). Antigen visualization was performed using an Envision Flex (Dako).

Pakuła M., Witucka A., Uruski P., Radziemski A., Moszyński R., Szpurek D., Maksin K., Woźniak A., Sajdak S., Tykarski A., Mikuła-Pietrasik J, & Książek K. (2019). Senescence-related deterioration of intercellular junctions in the peritoneal mesothelium promotes the transmesothelial invasion of ovarian cancer cells. Scientific Reports, 9, 7587.

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Immunohistochemical Characterization of Peritoneal Xenografts

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Formalin-fixed, paraffin-embedded sections from the peritoneal xenografts were examined as previously described [10 (link)]. Visualization was achieved using the EnVision + peroxidase system (Dako, Glostrup, Denmark). The antibodies used were against EpCAM and MUC4 (Abcam, Cambridge, UK), as well as Ber-EP4, carcinoembryonic antigen (CEA), calretinin, WT-1, CK20, CK7 and CDX2 (Dako). In addition the antibodies PAX8 (Abnova, Taipei City, Taiwan), B72.3 (BioGenex, Fremont, CA), Claudin-3 (CLD3) (Zymed, San Fransisco, CA), and Villin (Immunotec, Indianapolis, IN) were used. Negative control consisted of sections that underwent similar staining procedures with nonrelevant rabbit immunoglobulins or a monoclonal antibody of the same isotype as the primary antibody used. For all antibodies, positive controls were included with satisfactory results. The study pathologist (BD) evaluated immunohistochemical staining.

Andersson Y., Haavardtun S.I., Davidson B., Dørum A., Fleten K.G., Fodstad Ø, & Flatmark K. (2017). MOC31PE immunotoxin – targeting peritoneal metastasis from epithelial ovarian cancer. Oncotarget, 8(37), 61800-61809.

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Comprehensive Immunohistochemical Staining Protocol

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Immunohistochemical staining was performed in the Johns Hopkins Immunopathology Laboratory on formalin-fixed, paraffin-embedded tissue sections, using Ventana Benchmark automation and the Ultra View detection kit (Ventana Medical Systems, Tucson, AZ, USA) as previously described [26 (link),27 (link)]. All immunostains were performed at the time of diagnosis for primary or recurrent tumor. Antibodies from Cellmarque, Hot Springs, AZ, included: c-Kit (YR145), DOG-1 (SP31), CAM5.2 (B22.1&B23), Calretinin (Polyclonal), Cyclin D1 (SP4); antibodies from Ventana, Tucson, AZ, USA, included: CD34 (QBE/10), CD68 (KP-1), SMA (1A4), AE1/AE3 (pck-26), Actin (MOUSE), S100 (4C4.9), ER (SP-1), Melan A (A103); antibodies from DAKO, Carpinteria, CA, USA, included Desmin (D33) and Caldesmon (h-CD); antibodies from Abcam, Cambridge, MA, included ALK (5A4), Cathepsin K (3F9), and SDHB (21A11AE7); antibodies from Leica, Bannock Burn, IL, USA, included CD10 (MS/56C6) and PR (1E2); the rest of the antibodies included SOX-10 (N-20, Biocare, Concord, CA, USA), HMB-45 (HMB45, Novocastra, Newcastle upon Tyne, UK), Inhibin (R1, Serotec, Raleigh, NC), STAT6 (D-1, Santa Cruz, Santa Cruz, CA, USA), and SF-1 (n1665, Invitrogen, Waltham, MA, USA).

Liu Y., Shahi M., Miller K., Meyer C.F., Hung C.F., Wu T.C., Vang R, & Xing D. (2022). Gastrointestinal Stromal Tumors Mimicking Gynecologic Disease: Clinicopathological Analysis of 20 Cases. Diagnostics, 12(7), 1563.

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Comprehensive Immunofluorescence Staining Protocol

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Immunofluorescence experiments were performed essentially as described^{5 ,10 (link)}. Antibodies used include Calbindin (1:1,000, Swant, 300), Calretinin (mouse, 1:500, DAKO, M7245), Calretinin (mouse, 1:1,000, Swant, 6B3), Somatostatin (rabbit, 1:5,000, Bachem, T-4103.00), NKX2-1 (mouse, 1:300, Invitrogen, 18-0221), ISLET1 (mouse, 1:100, DSHB, 39.3F7), Pan DLX (rabbit, 1:1,000, gift from Y.M. Morozov, Department of Neurobiology, Yale University School of Medicine), NeuN (mouse, 1:100, Millipore, MAB377), GAD1 (mouse, 1:1,000, Millipore, MAB5406), GAD2 (mouse, 1:500, DSHB, GAD6), Synapsin-1 (rabbit, 1:1,000, Synaptic System, 106-002), DCX (rabbit, 1:1,000, Cell Signaling, 4604S), OLIG2 (rabbit, 1:1,000, Millipore, AB9610), TUJ1 (mouse, 1:1,000, Covance, MMS-435P), TUJ1 (rabbit, 1:1,000, Covance, MRB- 435P), VGAT (mouse, 1:1,000, Synaptic System, 131 003), Human nuclei antigen (mouse, 1:100, Millipore, MAB1281), GFP (chicken, 1:1,000, Aves Labs, GFP-1020), COUPTFII (mouse, 1:300, Perseus Proteomics, PP-H7147-00), MAP2 (chicken, 1:20,000, Abcam, AB5392), MAP2 (mouse, 1:500, Sigma Aldrich, M4403), GABA (rabbit, 1:1,000, Sigma Aldrich, A2052), Parvalbumin (mouse, 1:1,000, Sigma, P3088) (see also Supplementary Table 1).

Yang N., Chanda S., Marro S., Ng Y.H., Janas J.A., Haag D., Ang C.E., Tang Y., Flores Q., Mall M., Wapinski O., Li M., Ahlenius H., Rubenstein J.L., Chang H.Y., Buylla A.A., Südhof T.C, & Wernig M. (2017). Generation of pure GABAergic neurons by transcription factor programming. Nature methods, 14(6), 621-628.

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Immunohistochemical Analysis of Lung Biopsies

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Lung biopsies were obtained during redo-transplantation, VATS-biopsy or autopsy of patients with BOS (n=19) and RAS (n=19). Non-transplant control biopsies (n=14) were obtained during autopsy of patients without underlying lung disease (n=11) or during resection of non-diseased lung tissue from lung carcinoma patients with normal spirometry (n=3). All biopsies were retrieved at random by the Hospitals Pathology Department for routine clinical purposes and not specifically for this study. Paraffin sections were prepared from each biopsy and stained with calretinin (Dako, Agilent, Heverlee, Belgium) and/or TGF-β1 (Atlas, Bromma, Sweden) antibody (supplementary method 2 & 3). Control stainings for TGF-β1 were performed on liver and tonsil biopsies.

Sacreas A., von der Thüsen J.H., van den Bosch T.P.P., Weynand B., Verbeken E.K., Debbaut C., Van Raemdonck D.E., Vos R, & Verleden S.E. (2019). The pleural mesothelium and transforming growth factor-β₁ pathways in restrictive allograft syndrome: A pre-clinical investigation. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation, 38(5).

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