By Patrick Legembre
This quantity presents the main time-honored tools and protocols to review the apoptotic and non-apoptotic roles of CD95. Chapters discover molecular, biochemical, mobile tools and animal types to for you to larger comprehend the organic capabilities of this cytokine. Written within the hugely winning Methods in Molecular Biology series layout, chapters comprise introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, simply reproducible laboratory protocols, and tips about troubleshooting and warding off identified pitfalls.
Authoritative and useful, CD95: tools and Protocols goals to foster unique learn on CD95/CD95L couple.
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Extra info for CD95: Methods and Protocols
25 μg/mL of mouse anti-human FADD (BD PharmingenTM 556402). Incubate cells with a volume of 50 μL for 20 min at 4 °C in a humidity chamber. 5 PLA Probes Aspirate the primary antibodies solution and wash slides with 50 μL of blocking solution. The selection of PLA probes depends on the species of the primary antibodies. Dilute all PLA secondary antibodies (1/5) in blocking buffer or in Duolink In Situ Antibody Diluent (DUO92102) according to the manufacturer’s instructions in a final volume of 40 μL.
Equilibrate for 20 min at room temperature before using. Incubate slides/coverslips with PLA probes in a preheated humidity chamber for 1 h at 37 °C. 6 Ligation Remove PLA probe solution by aspiration. Wash slides/coverslips twice with a volume of 50 μL of wash buffer (Duolink® In Situ Wash Buffer A). Dilute the Ligation stock (1/5) in high purity water and mix. Dilute the ligase 1/40 in the ligation solution to obtain a final volume of 40 μL and vortex. 46 Melissa Thomas and Patrick Legembre Add the ligase solution to each sample.
Estornes Y, Toscano F, Virard F, Jacquemin G, Pierrot A, Vanbervliet B, Bonnin M, Lalaoui N, Mercier-Gouy P, Pacheco Y, Salaun B, Renno T, Micheau O, Lebecque S (2012) dsRNA induces apoptosis through an atypical death complex associating TLR3 to caspase-8. Cell Death Differ 19(9):1482–1494. Lu M, Lawrence DA, Marsters S, AcostaAlvear D, Kimmig P, Mendez AS, Paton AW, Paton JC, Walter P, Ashkenazi A (2014) Opposing unfolded-protein-response signals converge on death receptor 5 to control apoptosis.