By Leonardo Salmena, Vuk Stambolic
This quantity offers specified descriptions of triumphing and novel recommendations utilized by specialists within the learn of PTEN functionality in illness and biology. The ebook starts with chapters exploring ways to notice expression degrees of PTEN in basic and diseased human specimens; the right way to assessment particular PTEN functionality in mind melanoma; equipment that make the most of a brand new biosensor to degree PTEN law; and strategies to degree post-transcriptional rules of PTEN via micoRNAs and ceRNAs. different chapters current equipment describing novel options to discover PTEN localization and formerly unstudied structural positive factors of PTEN measured via X-Ray Crystallography and Hydrogen Deuterium trade Mass Spectrometry. The e-book concludes with how you can examine PTEN functionality in version organisms together with mice and C. elegans. Written within the hugely successful Methods in Molecular Biology series structure, chapters comprise introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, comfortably reproducible laboratory protocols, and tips about troubleshooting and heading off identified pitfalls.
Authoritative and thorough, PTEN: equipment and Protocols is a beneficial number of methodologies and protocols invaluable to researchers who're attracted to the PTEN field.
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Additional info for PTEN: Methods and Protocols
26 Mireia Castillo-Martin et al. 20. 10 % Formalin solution. 21. Agarose. 22. Steamer. 23. Pretreatment buffer (pH 6: Citric buffer, pH 8–9: EDTA buffer). 24. Blank sections for positive control tissues with known expression of the target protein. 25. Cytoseal XYL (Richard-Allan Scientific, Kalamazoo, MI). 26. Wet chamber, or a plastic box with lid equipped with a moistened paper towel (see Note 1). 27. Fixatives for fresh tissue and cells (see Note 2). 28. 4',6-Diamidino-2-phenylindole, dihydrochloride (DAPI) in mounting medium.
HL-60 cells were established from the peripheral blood of a 35-year-old woman with acute myeloid leukemia (AML FAB M2 ). HL-60 cells resemble promyelocytes but can differentiate terminally in vitro in the presence of dimethylsulfoxide (DMSO) or retinoic acid to granulocytes . Other compounds like 1,25-dihydroxyvitamin D3, 12-O-tetradecanoylphorbol13-acetate (TPA), and GM-CSF can induce HL-60 to differentiate to monocytic, macrophage-like, and eosinophil phenotypes, respectively. 2. U937 are myeloid cells established from the pleural effusion of a 37-year-old male patient with histiocytic lymphoma .
1 IHC on Frozen Tissue Samples with Peroxidase We propose the following procedures to perform IHC staining in frozen sections. 1. Let the slides warm to RT and fix (see Note 2) for 10 min at RT. 2. Circle the tissue on the slide with a hydrophobic pen. 3. Apply 10 % blocking serum in antibody diluent for 45 min at RT (see Note 9). 4. Aspirate. 5. Apply primary antibody and incubate overnight at 4 °C. 6. Aspirate. 7. Rinse well in running tap water for 5 min at RT. 8. Wash in PBS for 5 min, three times at RT.