SO
Reviewer
Silvia Olivera-Bravo
  • Faculty, Clemente Estable Biological Reseach Institute
Research fields
  • Neuroscience
Development and Characterization of Primary Brain Cultures from Japanese Quail Embryos
Authors:  Shaden Zoabi, Achinoam Blau and Shai Berlin, date: 09/20/2024, view: 290, Q&A: 0

Cell cultures play a crucial role in neuroscience research, facilitating the elucidation of the complexities of cellular physiology and pathology. The relative simplicity in producing cultures and the accessibility to cells that the cultures provide, in contrast to in vivo settings, allow users to manipulate and monitor cells more easily at higher throughputs and lower costs. These are ideal for screening purposes and electrophysiological characterizations. Despite the prevalence of methodologies for producing brain cultures from various animal models, rodents in particular, approaches for culturing neurons (and glia) from birds are less established or completely absent as in the case of the Japanese quail model. Here, we present a unique culturing protocol for brain cells (e.g., neurons at different maturation levels, such as progenitor cells, excitatory and inhibitory neurons, microglia, and endothelial cells) from entire forebrains of Japanese quail embryos for high-throughput screening of viral vectors in vitro and other various purposes. Following dissection and digestion methods uniquely suited for avian brains, we tailored the growth media and culturing surface to allow the survival of quail brain cultures for more than three weeks in vitro.

Click-iT® Plus OPP Alexa Fluor® Protein Synthesis Assay in Embryonic Cells

This protocol describes a method to assess relative changes in the level of global protein synthesis in the preimplantation embryo using the Click-iT® Plus OPP Protein Synthesis Assays. In this assay, O-propargyl-puromycin (OPP), an analog of puromycin, is efficiently incorporated into the nascent polypeptide of newly translated proteins in embryonic cells. OPP is fluorescently labeled with a photostable Alexa FluorTM dye and detected with fluorescence microscopy. The intensity of the fluorescence is quantitatively analyzed. This is a fast, sensitive, and non-radioactive method for the detection of protein synthesis in early embryo development. It provides a tool for analyzing the temporal regulation of protein synthesis, as well as the effects of changes in the embryonic microenvironment, and pharmacological and genetic modulations of embryo development.


Graphical abstract:



Figure 1. Brief overview of the procedures of the Click-iT® Plus OPP Alexa Fluor® protein synthesis assay in embryonic cells. (A) Set up OPP treatments: (1) Set up microdrops containing 50 µL of OPP working solution and label different treatments on the back of culture dishes (e.g., T0, T1, T2, and T3); (2) The drops are overlain with 2–3 mm heavy paraffin oil and then equilibrated in incubator for 2 h; (3) Collect the embryos from female reproductive tracts or following in vitro culture in desired treatments; (4) Culture embryos in the equilibrated OPP working solution for 2–6 h. (B) Example of OPP detection procedures working with 60-well plates labeled as T0, T1, T2, T3, T4, and T5 for different treatments: (1) The first 60-well plate is used for the procedures of washing, fixation, permeabilization, and Click-iT® OPP detection. (2) The second 60-well plate is for DNA staining and washing. (C) Slide preparation: (1) Label the required number of slides and set up vaseline coverslip supports; (2) Add mounting medium; (3) Transfer embryos into mounting medium; (4) Set coverslip; (5) Seal the coverslip with nail polish.

Pericyte Mapping in Cerebral Slices with the Far-red Fluorophore TO-PRO-3

This protocol describes a method for high-resolution confocal imaging of pericytes with the far-red fluorophore TO-PROTM-3 Iodide 642/661 in cerebral slices of murine. Identification of pericytes with TO-PRO-3 is a short time-consuming, high cost-effective and robust technique to label pericytes with no need for immunostaining or generation of reporter mice. Since the TO-PRO-3 stain resists immunofluorescence, and lacks spectral overlap, the probe is well suited for multiple labelling. Our procedures also combine TO-PRO-3-staining of pericytes with fluorescent markers for astrocytes and vessels in brain slices. These approaches should enable the assessment of pericyte biology in gliovascular unit.

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