Scale bar, 20?m. (D) Representative image showing an over-fixed cortical slice. be replaced by sterile PBS. electroporation of mouse brains (major step 2 2) Step-By-Step Method Details Polarity Acquisition Major steps 1C2 Ibotenic Acid do not need sterile conditions. You will need a stereo-microscope to continue with the dissection. Tissue homogenization can be done using micropipettes (200C1,000?L). Repeat up-and-down movements until obtain a homogenous suspension, following the rule of using, first, a large tip (1,000?L) and then a small 1 (200?L). The goal is to isolate neurons gradually, avoiding cell clumps, without – or the less possible – mechanical stress. After plating, hippocampal and cortical neurons will transform from symmetric cells (stages 1C2; 0.5C1 DIV) to neurons with well-defined axonal and dendritic compartments (stages 3C5; 3C14 DIV (Dotti et?al., 1988; Kaech and Banker, 2006; Cceres et?al., 2012). Of notice, culture occasions indicated below are relevant to middle-low cell culture densities. It is important to spotlight that timing suggested below may vary depending on neuronal confluency after plating, culture media composition and covering (for extended feedback, please see problem 3 in troubleshooting). In addition, later stages (3C5) could be sub-divided (e.g., early and late-stage 3; 2 and 4 DIV, respectively). Currently, several molecules have been identified as markers, Ibotenic Acid enriched in either the axonal or dendritic domain name. Accordingly, Rabbit Polyclonal to IL4 Table 1 summarizes a battery of markers to identify these compartments during the acquisition of neuronal polarity. In addition, cytoskeleton dynamics, vesicle trafficking and Ibotenic Acid organelle distribution are instrumental to predict the axo-dendritic specification. The following table (Table 2) enlists events to be considered during the acquisition of neuronal polarity. Table 2 Cytoskeleton Dynamics, Vesicle Trafficking and Organelle Distribution Parameters Sustaining Axo-Dendritic Specification Electroporation of E15.5 Mouse Brains involves 4 main steps: anesthesia of the animal, in utero electroporation (IUE), post-electroporation procedures, and imaging. The expression of plasmids encoding fluorescent proteins, such as GFP, allows monitoring the migration and polarization of cortical neurons during embryonic corticogenesis (as well as in post-natal stages). In this protocol, we focus on E15.5-E17.5?days, because within this time-frame neurons undergo morphological transformations that resemble the first stages of polarity acquisition in culture (Kriegstein and Noctor, 2004; Barnes and Polleux, 2009; Takano et al., 2019). 6. Anesthesia and sterilization of the animal a. Place the pregnant mouse inside the chamber made up of the isoflurane?+ oxygen mix for anesthesia. The anesthesia process consists of two phases: i. Induction: 4% isoflurane circulation is needed to accomplish full anesthesia in short time (visible by a reduction on the breath frequency). ii. Maintenance: Place the animal in the warming system at 37C. Make sure to expose the mouth into the anesthesia device and set isoflurane circulation to 2%C2.5% for the surgery. To confirm full anesthesia, pinch the toes with a tweezer and confirm the lack of response. b. Make a subcutaneous injection of 1 1?mg/kg tramadol (or equivalent). c. Sterilize the animal: i. Shave the stomach using a razor knife or an electric razor. ii. Disinfect the zone with iodine answer and 70% v/v ethanol. 7. Embryo exposure a. Make and incision with fine scissors in the stomach, along the alba collection, of both skin and muscle mass layers. An incision of 5?mm length is enough to expose embryos (Physique?3; first step). Open in a separate window Physique?3 DNA Injection and IUE of Mouse Brains (E15.5) electroporation (IUE) of E15.5 mouse brains involves several steps, including anesthesia with isoflurane, embryo exposure, DNA injection & electroporation, recovery after surgery, histology, and imaging of GFP-positive brains. The plan summarizes.