C Programming With Arduino Warwick Pdf ((BETTER))
CLICK HERE ===> https://urluso.com/2t9AN9
Fourteen-week-old male C57BL/6J mice were used in all experiments. All surgeries were performed under aseptic conditions under isoflurane anesthesia. Mice were given postoperative analgesia in the form of buprenorphine and meloxicam or in some cases carprofen. Core body temperature was maintained at 37°C during surgery by means of a heating pad. All experiments were performed at the same time of day, with the exception of the experiment in Figure 3. A total of 2.5–2.8M neurons were electroporated in each experiment. In one experiment involving anesthesia (Figure 6), parabrachial nucleus injections were verified in animals that were anesthetized for 7–9 days after injection. Mice were euthanized and perfused with 4% paraformaldehyde (PFA) for immunohistochemistry. Brains were removed and fixed overnight in 4% PFA, cryoprotected in a 30% sucrose solution, and flash frozen. Transverse sections (30 μm) were cut with a cryostat and stored at −20°C until staining.
For experiments involving retrograde tracer injections, two different anterograde tracers were used: cholera toxin B (CTb) and biocytin. Both tracers were conjugated to Alexa 488, Alexa 594, or Alexa 647 (Life Technologies, 1:1000 dilution) and were injected at a concentration of 0.2% in phosphate buffered saline (PBS). For CTb injections, 2% wheat germ agglutinin–Alexa 488 (Life Technologies) was included in the injectate, as previously described.14 For biocytin injections, 1% Evans blue (Sigma-Aldrich) was included in the injectate. Pups were euthanized 4 days after injection and brains were fixed overnight in 4% PFA, cryoprotected in 30% sucrose, and flash frozen. Transverse sections (30 μm) were cut with a cryostat and stored at −20°C until staining. Sections were incubated with a mixture of primary and secondary antibodies diluted in PBS containing 0.1% Triton X-100 (1:200–1:1000) and mounted on microscope slides using Vectashield mounting media with DAPI (Vector Laboratories).
For bulk RNA extraction, brain tissue was isolated from mouse brain using TRIzol (Invitrogen). The dissected tissue was then dissociated in TRIzol by trituration using a P1000 pipette. The tissue was then left to sit at 4 °C for 10 min to allow for phase separation. The aqueous phase containing the RNA was collected and RNA was precipitated by the addition of isopropanol. The precipitated RNA was centrifuged at 12,000 rcf for 15 min at 4 °C. The RNA pellet was washed with 75% EtOH twice. The RNA pellet was then dissolved in H2O. RNA was quantified using a NanoDrop 2000. To obtain mRNA, the DNAse digestion was performed using Turbo DNAse (Ambion). The reverse transcription was performed using SuperScript III (Invitrogen) and the miR-124 specific reverse primer (Geneworks Biotechnologies). For the analysis of specific miRNA, primers were designed (miR-124-specific reverse primer, Geneworks Biotechnologies). The amplification of specific miRNA was performed using SYBR Green II PCR Master Mix (Applied Biosystems). The results were normalized against the small nuclear RNA U6. 827ec27edc