TSQ fluorochrome incubation increase autometallographic zinc detection in primary cell cultures of astrocytes

• Main Authors: Ballestín Hinojosa, Raúl, Torres, Jose Manuel, Ponsoda i Martí, Xavier
• Format: Conference Object
• Language: English
• Published: 2021
• Subjects: astrocito; cultivo celular; zinc; tsq; autometalografía; UNESCO::CIENCIAS DE LA VIDA; Olympus; Orca
• Online Access: https://hdl.handle.net/10550/82229

In this work, we have analyzed the combination of direct (TSQ) and indirect (AMG) zinc detection methods in order to increase the sensitivity to a better study of the zinc uptake. For that purpose, rat primary cell cultured astrocytes were used. Intracellular Zn levels were visualized by using the TSQ zinc fluorochrome, either in normal conditions or after supplementary addition of 50 µM ZnSO4 to the culture. Fluorescence was recorded with an Olympus microscope BX50WI, equipped with a Hamamatsu ORCA digital camera controlled with the Aquacosmos software. Monolayers were fixed and ionic zinc precipitated by sodium sulphide or sodium selenite. Autometallographic zinc detection was the last step to detect zinc in cell cultures. Zincosome formation were visualized meanwhile the zinc fluorochrome TSQ in light microscopy, with or without adding zinc to cell monolayers. Additionally, we tested the better zinc precipitation and fixative method in order to preserve zinc ions and make them suitable for AMG development, selecting 0.1% sodium sulphide and 4% PFA. TSQ incubation demonstrated that are crucial for AMG zinc detection in both, prior and after the cell fixation. We found the setting parameters by which the zinc ion content of zincosomes can be maintained and visualized in optimal conditions and circumstances, either for optical and electron microscopy zinc detection studies.