In order to selectively label NAc- (mPFCNAc) and VTA-projecting mPFC cells (mPFCVTA), we injected Canine adenovirus type 2 carrying Cre-recombinase gene (CAV2-Cre, CMV promoter, titer: 2.5 × 1010 pp/ml, Plateforme de Vectorologie de Montpellier, France; a gift from D. Zelena) into the NAc (n = 3 animals) or VTA (n = 3 animals) (see coordinates above) of wild-type animals, mixed with 5% biotinylated dextrane amine (BDA, MW: 10.000, Molecular Probes: D1956, RRID: AB_2307337 ; 1:1; 80–120 nl/animal; 1 nl/s). Note that BDA was used to locate the tip of the injecting pipette (Figure 5B1, C1 ), not the whole extent of viral diffusion. At the same time, the mPFC (see coordinates above) of the same animals was injected with AAV5-EF1a-DIO-mCherry (see details above). After 6 weeks of survival, animals were perfused, and their brains were processed for further analysis (see above).
D 1956
Manufactured by Thermo Fisher Scientific
Sourced in France
The D-1956 is a laboratory instrument manufactured by Thermo Fisher Scientific. It is designed for general laboratory use and performs core functions related to sample analysis and preparation. The detailed specifications and intended applications of this product are not available in this response.
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Lab products found in correlation
Tyramide signal amplification fluorescence system, by PerkinElmer (1 mentions)
Streptavidin fitc, by Merck Group (1 mentions)
Eclipse ci, by Nikon (1 mentions)
Nanoject 2, by Drummond (1 mentions)
Kds310, by KD Scientific (1 mentions)
Stereotaxic instrument, by RWD Life Science (1 mentions)
Vibration microtome, by Leica camera (1 mentions)
D1817, by Thermo Fisher Scientific (1 mentions)
Stereotaxic apparatus, by RWD Life Science (1 mentions)
13 protocols using d 1956
1
Tracing mPFC Projections to NAc and VTA
2
Retrograde and Anterograde Tracer Injection in Mice
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Mice were anesthetized with (i.p., 5 mL/kg) injection of ketamine (87 mg/kg) and xylazine (13 mg/kg). 0.1 mL of local anesthetic 1% lidocaïne was subcutaneously injected under the scalp before placing the animal in the stereotaxic frame. A hydrating gel (Ocryl-gel) was regularly applied on the eyes of the animal to avoid dehydration. An incision was made in the scalp and a unilateral hole was drilled in the skull at the appropriate coordinates determined using the 4 th edition of the Mouse Brain Atlas (Paxinos and Franklin 2012) are listed in table 1. A glass micropipette (20-30 μm tip diameter) containing the retrograde tracer cholera toxin subunit B (CTB; 0.25 % in 0.1 M Tris and 0.1 % NaCl; Sigma, France Cat:C9903) or the anterograde tracer biotin dextran amine (BDA) at 2% in 0.9% NaCl (10,000 MW) (Molecular Probes D1956) was lowered into the target area. The tracer was ejected by iontophoresis using constant current source (Midgard; 3 μA, 7 sec on/off intervals) during 8±1 min for CTB or during 12±1 min for BDA. After microinjection, the pipette was left in place for 10 min and then removed slowly. The incision was closed with two fastenings and the animal was left in a clean cage under a warming lamp until awakening. It was then transferred to the home cage in the animal facility under daily monitoring with Metacam (2 mg/kg s.c.) injected daily for 3 days.
3
Tracing Descending CST Fibers in Grafted Mice
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Six weeks after injury, descending CST fibers of grafted mice were labeled with BDA (0.1 g/ml in sterile normal saline, Thermo-Fisher Scientific # D1956) by injection into five spots of the right motor cortex. The skulls of anesthetized mice were tightly fixed to a stereotaxic apparatus (Kopf, Tujunga, CA). Craniotomy over the motor cortex area was carried out using a micromotor system (Foredom, Bethel, CT). The injection area on the right hemisphere was defined in a rectangle with one side measuring 2 mm (from 1.0 mm anterior to −1.0 mm posterior to the bregma) and one side measuring 1.5 mm (lateral to the bregma). Injections were performed using a glass capillary attached to a microsyringe (Hamilton, Reno, NV) at a 0.7 mm depth. Each injection delivered 75 nl of BDA solution into the motor cortex at a rate of 75 nl/min. The tip of the glass capillary was left in place for two additional minutes before withdrawal. Two weeks later, the animals were sacrificed by transcardial perfusion with PBS followed by 4% formaldehyde. To visualize the BDA, a tyramide signal amplification fluorescence system (Perkin Elmer, Waltham, MA) was used.
4
Tracing Corticospinal Tract Axons in Mice
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On day 28 or 42 after SCI, mice were anesthetized, and their heads were fixed on a stereotaxic frame, and the corresponding skull area was carefully exposed. To label the CST axons, we injected 10% BDA solution (10,000 MW, D1956, Thermo Fisher Scientific) into the hindlimb region of the cerebral motor cortex using a glass capillary attached to a microsyringe (coordinates from bregma: 0.5 mm posterior/0.5 mm lateral, 0.5 mm posterior/1.0 mm lateral, 1.0 mm posterior/0.5 mm lateral, 1.0 mm posterior/1.0 mm lateral, at a depth of 0.5 mm, 0.5 μL each). The mice were killed 2 weeks after BDA injection, and spinal cord tissues were harvested.
5
Tracing Axon Regeneration after ICH
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At 21 d after ICH, 2 μL of BDA (10%, w/v, dissolved in PBS, D1956, Thermo Fisher Scientific) were injected into the contralateral cortex at two sites (x1 = 0.6 mm, y1 = 1.2 mm, z1 = 1.5 mm; x2 = 0.0 mm, y2 = 1.8 mm, z2 = 1.7 mm). 14 d later, slices were collected at the levels of facial nucleus (FN) and 7th segment of cervical spinal cord (C7) (35 μm thick), and incubated with Streptavidin-FITC (S3762, Sigma) to label BDA+ axons. Images were obtained by a fluorescence microscope (Nikon ECLIPSE Ci). Three sections at each level were selected for each mouse. The number of midline-crossing BDA+ fibers was quantified [29 (link),30 (link)].
6
BDA Tracer Injection in V2 Cortex
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A craniotomy above the right V2 was made 4.0–5.0 mm posterior from the bregma, 2.0–3.0 mm lateral of the midline [105 ]. A glass capillary (tip 10–20 μm) filled with 10% BDA (D1956; Thermo Fisher Scientific, Waltham, MA) in 0.1 M phosphate-buffered saline (PBS) was installed with an auto-nanoliter injector (Nanoject II; Drummond Scientific, Broomall, PA) and then inserted into the V2 according to the following 4 stereotaxic coordinates: (in mm) AP −4.50, ML 2.25; AP −4.50, ML 2.75; AP −5.00, ML 2.25; AP −5.5, ML 2.25. For each coordinate, 0.3 μl of the BDA solution was ejected at 0.7, 0.85, and 1 mm below the pia at a rate of 0.09 nl/s. After ejections, the capillary tip was held in the position for at least 5 min and then gently retracted. The craniotomy was covered with sterile bone wax, and the skin wound was sutured. After 1 wk of survival, the rats were deeply anesthetized and perfused, and the brains were processed (see section Tissue processing and immunohistochemistry).
7
Tracing Axonal Sprouting After Stroke
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Biotinylated dextran amine (BDA, D1956, Thermo Fisher Scientific) was injected into the ipsilateral side of the infarcted cortex to trace axonal sprouting from the rostral forelimb area (RFA) following ischemic stroke. It has been reported that the RFA is a frequent region where functional reorganization occurs after a stroke in the CFA [21 (link)-23 (link, link)]. Functional reorganization after CFA stroke gradually occurred in RFA up to 4 weeks [24 (link)]. Therefore, BDA injection was performed at 4 weeks after the stroke induction. After animals were placed on a stereotaxic frame, a 1 mm-diameter burr hole was made on the skull using a hand drill (Fig. 2A ). After loading one μl of 10% BDA to a pulled-capillary glass on a Hamilton syringe, BDA was delivered into the cerebral cortex at the predetermined coordinate of the RFA (AP: +1.5 mm; ML: -1.8 mm; DV: -1.0 mm) using an infusion pump (KD Scientific, KDS310) at a rate of 0.4 μl/min. The capillary glass needle was removed 2 min after the infusion was completed. The incision wound was sutured and sterilized.
8
Anterograde Tracing of Dorsal and Ventral Hippocampal CA1
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After the mice were anesthetized with ketamine and xylazine (160 mg/kg and 12 mg/kg body weight, respectively) and secured in the stereotaxic instruments (RWD, Shenzhen, China), anterograde tracing marker, biotinylated dextran amine (BDA) (10,000 MW, D-1956; Invitrogen; 10% dissolved in 0.1 M PH 7.4 phosphate buffer), was injected into the CA1 of dorsal hippocampus and the CA1 of ventral hippocampus (AP, −3.16; ML, ±3.8; DV, −4.1) based on the atlas of Franklin et al. (2008) in a volume of 0.3 μl and retained in place for an additional 10 min to optimize diffusion. Following the BDA tracing in axons for 7–10 days, mice were perfused with 0.9% saline and followed by 4% paraformaldehyde in phosphate buffer (PFA, pH 7.4). Then, the brains were removed and post-fixed in 4% PFA for 18–24 h. All brains were cut in 40 μm coronal sections on a vibration microtome (Leica) and collected in 0.01 M PBS. The mice with a wrong injection site were not included in data analysis.
9
Axon Tracing in Motor Cortex Post-Pyramidotomy
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Axon tracer injection was performed essentially as described previously [4 (link)]. In brief, 0.6 μl of 10% biotinylated (Invitrogen, D1956) or rhodamine-conjugated dextran amine (Invitrogen, D1817) in phosphate buffered saline was injected into three points of the cerebral cortex (1 mm right/0 mm anterior, 1 mm right/0.5 mm anterior, and 1.5 mm right/0.5 mm anterior to the bregma, depth = 0.6 mm, total = 1.8 μl). This area corresponds to the forelimb area of the motor cortex [57 (link)]. Injection was performed 2 weeks after pyramidotomy and mice were sacrificed 2 weeks after injection (4 weeks after pyramidotomy).
10
Stereotaxic Dye Injections in Mice
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Mice were anesthetized and mounted on the stereotaxic frame. Bilateral injections of the biotin or Texas red dextran amines (5% solution, D-B, D-1956 or D-TRD1863, Invitrogen) were performed at different rostro-caudal levels of the DMV: AP −7.8 mm, −7.9 mm and −8.0 mm. The stereotaxic coordinates used for lateral ventricle injection were AP −0.5 mm, L −1.0 mm and V −2.0/−1.50/−1.0 mm. Injections (4 ms duration) were performed using a glass micropipette (25 µm). At the end of the injection procedure, the wound was closed by a suture with Mersilene, 6–0 silk.
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