immunolabeling of neuronal membrane proteins in a freeze-fractured specimen of mouse brain tissue

Immunolabeling of Neuronal Membrane Proteins in a Freeze-fractured Specimen of Mouse Brain Tissue

For SDS digestion, transfer a replica to a 4 milliliter glass vial filled with 1 milliliter of SDS-digestion buffer. Allow it to digest for 18 hours at 80 degrees Celsius with shaking. For immunolabeling, wash the replica for 10 minutes in fresh SDS-digestion buffer. Then, incubate it with primary and secondary antibodies diluted in 2% BSA TBS in a humid chamber at 15 degrees Celsius for 24 to 72 hours.After that, mount the replica on a formvar-coated, 100-line parallel bar grid. Image the replica with a transmission electron microscope at 80 or 100 kilovolts. Then acquire the digital images through a CCD camera.

immunolabeling-neuronal-membrane-proteins-freeze-fractured-specimen

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.&#160; &#160;1. Specimen Preparation<ol><li>Brain fixation<ol style='list-style-type:decimal;'><li>Preparation of the fixative<ol style='list-style-type:decimal;'><li>For 1 L of fixative, weigh 10 g of paraformaldehyde and add it to 300 ml of deionized water (H2O). Heat to 55 - 60 &#186;C for ~10 min with continuous stirring.</li><li>Switch off the heat and add 7 - 8 drops of 4 N Sodium Hydroxide (NaOH). The solution should become clear in ~10 minutes.</li><li>Let it cool down to room temperature or RT, add 150 ml of a saturated solution of Picric acid, and bring it to 500 ml with deionized H2O.</li><li>Add 500 ml of 0.2 M phosphate buffer (PB). Filter with filter paper. Adjust pH to 7.4 with NaOH.</li><li>Cool the fixative to 6 &#186;C, and store it in dark glass bottles for not more than one day at 6 &#186;C.</li></ol></li><li>Transcardiac perfusion<ol style='list-style-type:decimal;'><li>Anesthetize mice with an intraperitoneal injection of thiopental (120 mg/kg body weight). Make sure the animal is deeply anesthetized by checking the pedal withdrawal reflex, which should be absent. Place the animal on its back on a perfusion table with the four extremities tied down.</li><li>Open the abdominal wall longitudinally with blunt-end scissors and make two additional cuts laterally along the caudal border of the rib cage to expose the diaphragm. Cut away the diaphragm and cut the thoracic wall at the osteocartilageneous border on both sides. Lift the caudal end of the central slab of the thoracic wall containing the sternum to expose the heart.</li><li>Remove the pericardium and make a small, precise cut in the tip of the left ventricle to admit the cannula of the perfusion apparatus. Use a blunt cannula with an internal diameter of 0.6 mm. Pass the cannula gently through the ventricle till the tip appears into the ascending aorta, and secure the cannula with a clamp. To allow the blood and perfusates to exit from the blood stream, make a cut in the right atrium.</li><li>Perfuse mice transcardially using a peristaltic pump at a flow rate of 5 ml/min at first with phosphate buffer saline or PBS (25 mM, 0.9% sodium chloride or NaCl, pH 7.4) for approximately 1 min, followed by ice-chilled fixative for 7 min.</li><li>After fixation, sever the mouse head with a pair of scissors and then cut the skin through the midline from neck to nose. Remove the muscle to expose the skull fully.</li><li>Using sharp scissors, make a longitudinal cut through the occipital and interparietal bones starting from the foramen magnum. Using fine tweezers remove these bones to expose the whole cerebellum. Then make another longitudinal cut through the parietal and frontal bones till the nasal bone and remove them with tweezers to expose the whole brain.</li><li>Using a spatula, remove the brain without damaging it and place it in ice-cold 0.1 M PB.</li></ol></li></ol></li><li>Sectioning and trimming of the specimens<ol style='list-style-type:decimal;'><li>Cut a coronal block of approximately 5 - 6 mm with razor blades containing the area of interest. Glue it onto the holder of the vibroslicer with a cyanoacrylate glue. Orient the tissue block so that the neocortex faces the vibrating blade. Slice coronal sections containing the amygdala at 140 &#181;m with the vibroslicer (Figure 1A) in ice-cold 0.1 M PB and collect them in a 6-well dish in the same buffer.</li><li>Under a stereomicroscope, trim out the region of interest (here, the medio-dorsal paracapsular cluster of the intercalated cell masses of the amygdala or ITC; see Figure 1B) from the slice. Do this in a Petri dish coated with silicone elastomer and filled with 0.1 M PB, using an ophthalmic scalpel. Make sure that the trimmed blocks fit into the hole of the spacer (approximately 1.5 mm) (Figure 1B).</li><li>Move the trimmed blocks into cryoprotection solution (30% glycerol in 0.1 M PB) overnight (O/N) at 6 &#186;C.</li></ol></li></ol>2. High-Pressure FreezingNOTE: The Freeze-fracture Replica Immunolabeling (FRIL) consists of 6 essential steps (Figure 2): 1) Rapid freezing with high pressure (at 2,300 - 2,600 bar) of the specimen. 2) Fracturing of the specimen. The fracture plane generally follows the central hydrophobic core of frozen membranes, splitting them into two half-membrane leaflets: a half that lies adjacent to the protoplasm (P-face) and a half that lies adjacent to the extracellular or exoplasmic space (E-face). 3) Replication of the specimen by vacuum-deposition of platinum and carbon. 4) Detergent-digestion of the tissue. 5) Immunogold labeling. 6) Analysis of the replica using a transmission electron microscope.<ol><li>Preparation of copper carriers &#160;NOTE: To be handled through the successive stages of the FRIL procedure, specimens need to be mounted onto metal (gold or copper) carriers. These carriers vary in size and design according to the mode of fracturing and the type of machines used. Here, we used copper carriers (Figure 1B, E) and a hinged "double replica table" (see Figure 3B), which, when opened, produces a tensile fracture through the frozen specimen (Figure 2). This allows to retain and replicate both sides of the fractured specimen.<ol style='list-style-type:decimal;'><li>Polish copper carriers with a tarnish remover using a sheet of chamois skin.</li><li>Place carriers in a glass pot and clean twice with a non-ionic detergent (pH ~1.5) in a sonicating water bath, then extensively wash in tap water followed by deionized water, and then rinse twice with ethanol.</li><li>Sonicate the copper carriers in acetone for 15 min.</li><li>Place the carriers on filter paper to dry.</li><li>Attach a ring of double-sided tape to a copper carrier (Figure 1C), which will serve as the holding well for the trimmed block (holding carrier).</li></ol></li><li>Freezing of the specimen &#160;NOTE: Handle liquid nitrogen with care and wearing appropriate goggles.<ol style='list-style-type:decimal;'><li>Turn on the high-pressure freezing unit (Figure 1F) for at least 1.5 hours before freezing the specimen.</li><li>Start heating by pressing the "AIR HEATER" button, and bake out for 50 min. Set air temperature to 80 &#176;C.</li><li>Connect the nitrogen tank to the high-pressure freezing unit and press the "NITROGEN" button to fill the inside Dewar with liquid nitrogen. The "NITROGEN LEVEL" lamp lights up. Start cooling by pressing the "Cooling" button.</li><li>Press the "DRIVE IN" button when the "NITROGEN LEVEL" goes out. Check that the hydraulic system moves the piston back and forth 3 times.</li><li>Press the "AUTO" button, and the "NITROGEN" button. As soon as "READY" lights up, the high-pressure freezing unit is ready for high-pressure freezing.</li><li>Place a trimmed block in the hole of the double-sided tape (Figure 1B) using a platinum wire loop which has been melted into a glass pipette.</li><li>Remove the excess of the cryoprotectant solution using filter paper or a brush. &#160;NOTE: This procedure is also important to remove air bubbles that may form around the tissue and which could cause distortion of the tissue shape and/or ultrastructure.</li><li>Under a stereomicroscope, cover the holding carrier with another carrier, so that the tissue block is sandwiched between the two carriers.</li><li>Insert the carrier sandwich into the specimen holder of the high-pressure freezing unit (Figure 1D). Insert the specimen holder into the high-pressure freezing unit (tip down) and secure it by screwing in the specimen holder.</li><li>Initiate the freezing cycle by pressing the "Jet-Auto" button. Working as quickly as possible, remove the specimen holder and submerge the tip with liquid nitrogen into an insulated box. Immerse the tips of two pairs of forceps in the liquid nitrogen to cool them.</li><li>Carefully remove the carrier-sandwich from the specimen holder and place it in a pre-chilled cryovial. Make sure that the carriers are only handled with liquid nitrogen-cooled forceps. Cryovials should be perforated to allow the nitrogen to flow out from the vial (Figure 1G).</li><li>Repeat steps 2.2.6 to 2.2.11 until all desired samples have been frozen. Multiple carrier-sandwiches containing the same type of sample can be stored in the same vial.</li><li>Store the cryovials containing the carriers in a cryotank until replication (Figure 1H).</li></ol></li></ol> &#160;3. Freeze-fracture and Replication<ol><li>Preparation of the electron beam guns<ol style='list-style-type:decimal;'><li>Before inserting the electron beam guns, remove the shield with the "deflector plate". Place the "setting gauge" to center the filament into the collet chuck through the lower cathode cover. &#160;NOTE: The larger diameter end of the setting gauge is used for the carbon gun, whereas the smaller diameter end is for the platinum gun.</li><li>Slide the new filament over the gauge until "the pressure laminae" can clamp the ends of the filament, ensuring that the filament coil does not lie at an angle.</li><li>Remove the setting gauge and insert the carbon rod. Fix it by tightening the collet chuck of the evaporator rod holder, ensuring that the height of the end of the rod is at the middle of the second coil from the bottom. For the platinum gun, the height of the end of the platinum rod should be at the middle of the second filament coil from the top.</li><li>Replace the deflector plate and insert guns into the freeze fracture unit. Clean the guns with a sand-blaster after usage.</li></ol></li><li>Set up of freeze-fracture unit<ol style='list-style-type:decimal;'><li>Switch on the freeze fracture unit (Figure 3A) by turning MAINS to 1. For a detailed description of the freeze-fracture and replication procedures, see the operating instructions provided by the manufacturer.</li><li>Prior to cooling the freeze-fracture device, bake out the entire cooling system of the unit with warm air. Press the "Thawing" button in the MTC 010 device (temperature control unit) (Figure 3A) and let the bake out process run for 45 min.</li><li>Activate the vacuum station. The freeze-fracture unit usually operates in a vacuum range of ~10-6&#160;- 10-7 mbar.</li><li>Fill nitrogen tank and connect it to the freeze fracture unit. Check that the valve holder is dry and also clean the entry of the tank before insertion of the valve holder (humidity can interfere with vacuum and indication of N2 filling of the tank).</li><li>Start cooling by setting the temperature to -115 &#176;C. Cooling takes about 45 min.</li><li>Insert the electron beam guns and adjust current and voltage to reach the following parameters for evaporation: &#160;Carbon gun: rotation on, position 90&#176;, rate of carbon accumulation 0.1 - 0.2 nm/sec &#160;Carbon-platinum gun: rotation off, position 60&#176;, rate of accumulation 0.06 - 0.1 nm/sec &#160;NOTE: If a gun is used for the first time after exchange of the carbon or platinum rod, degas for 3 min before usage.</li></ol></li><li>Fracturing and replication<ol style='list-style-type:decimal;'><li>Insert frozen carrier sandwiches into a double replica table making sure all manipulations are done in liquid nitrogen.</li><li>Transfer the double replica table to a Dewar vessel and fix it to the specimen stage receiver at an angle of 45&#176;. The liquid nitrogen level should always be above the double replica table.</li><li>Pick up the double replica table with the table manipulator and insert it into the freeze fracture unit onto the cold stage. Wait approximately 20 min to allow the temperature of the double replica table to adjust to -115 &#176;C.</li><li>Check that the vacuum is below 10-6 mbar and the temperature is -115 &#176;C.</li><li>Fracture the tissue by manual counter clockwise rotation of the wheel connected to the shroud placed above the double replica table. When the shroud turns, it forces the double replica table to open, fracturing the tissue.</li><li>Press the "High tension" button in the EVM 030 device (electron beam evaporation control unit) of the freeze-fracture unit (Figure 3A).</li><li>Replicate the exposed surfaces of the fractured tissue (Figure 3C) by evaporation of carbon (rotating) by means of an electron beam gun positioned at a 90&#176; angle to a thickness of 5 nm, followed by a unidirectional shadowing with platinum-carbon at a 60&#176; angle to a thickness of 2 nm. Finally, apply a 15 nm thick layer of carbon from a 90&#176; angle (rotating).</li><li>Use the following parameters for evaporation: &#160;1st carbon: rotation on, position 90&#176;; speed 0.1 - 0.2 nm/sec; 5 nm &#160;2nd carbon-platinum: position 60&#176;; speed 0.06 - 0.1 nm/sec; 2 nm &#160;3rd carbon: rotation on, position 90&#176;; speed 0.3 - 0.5 nm/sec; 15 nm</li><li>Remove the replicated specimens from the freeze fracture unit and transfer them to a ceramic 12-well plate (Figure 4A) filled with TBS (Tris-buffered saline, pH 7.4).</li><li>Using a platinum loop wire rod, remove the replicated tissue from the specimen carrier (Figure 4A).</li><li>Repeat steps 3.3.1 to 3.3.10 until all samples have been replicated.</li></ol>4. SDS-digestion of the replica<ol style='list-style-type:decimal;'><li>Transfer the replica to a 4 ml glass vial filled with 1 ml of SDS-digestion buffer (2.5% Sodium Lauryl Sulfate, 20% sucrose in 15 mM Tris, pH 8.3). Digest for 18 hr at 80 &#176;C with shaking (45 strokes/min).</li><li>Transfer replicas to a new tube filled with SDS-digestion buffer and store at RT.</li></ol></li></ol>4. ImmunolabelingNOTE: All incubations are performed at RT with gentle shaking except for incubations with antibodies.<ol><li>Wash the replica for 10 min in fresh sodium dodecyl sulfate (SDS)-digestion buffer.</li><li>Wash the replica once with 2.5% BSA (bovine serum albumin) in Tris-Buffered Saline or TBS for 5 min, and then 3 x 10 min with 0.1% BSA in TBS.</li><li>Block non-specific binding sites in TBS with 5% BSA for 1 hr.</li><li>Apply primary antibodies diluted in 2% BSA-TBS. Perform incubations in a 30 &#181;l drop (Figure 4B) in a humid chamber at 15 &#176;C for 72 hr (Figure 4C).<ol style='list-style-type:decimal;'><li>For this study, process both replicas from the fractured tissue. Incubate one replica with a guinea pig polyclonal antibody raised against the amino acids 717 - 754 of the mouse GluR1 common to all &#945;-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptor or AMPA-R subunits (dilution: 1:200) or a mouse monoclonal antibody raised against a recombinant fusion protein covering amino acids 660 - 811 of the NR1 subunit of the N-Methyl-D-Aspartate Receptor or NMDA-R (dilution: 1:500), and a rabbit polyclonal antibody raised against the green fluorescent protein (dilution: 1:300).</li><li>Incubate the other replica with a rabbit polyclonal antibody raised against a synthetic peptide corresponding to amino acids 384 - 398 of the rat &#181;-opioid receptor (dilution: 1:500).</li></ol></li><li>Wash in TBS with 0.05% BSA (3 x 5 min.).</li><li>Apply secondary antibodies. For this study, use gold (5 nm for ionotropic glutamate receptors, 10 for &#181;-opioid receptors, and/or 15 nm for Channelrhodopsin-2 Yellow Fluorescent Protein or ChR2-YFP) conjugated antibodies diluted in TBS with 2% BSA. Dilute secondary antibodies 1:30 and incubate in a 30 &#181;l drop at 15 &#176;C O/N.</li><li>Wash 3 x 5 min in 0.05% BSA-TBS at RT.</li><li>Wash 2 x 5 min in ultrapure water.</li><li>Mount replica on formvar-coated 100-line parallel bar grid (Figure 4D).</li></ol>

<figure class='table' style='width:470pt;'><table class='ck-table-resized'><colgroup><col style='width:32.55%;'><col style='width:34.89%;'><col style='width:32.56%;'></colgroup><tbody><tr><td style='height:14.5pt;width:470pt;' colspan='3'>Tissue preparation</td></tr><tr><td style='height:14.5pt;width:153pt;'>Paraformaldehyde EM grade</td><td style='width:164pt;'>Agar Scientific Ltd., United Kingdom</td><td style='width:153pt;'>AGR1018</td></tr><tr><td style='height:14.5pt;width:153pt;'>Saturated picric acid solution</td><td style='width:164pt;'>Sigma-Aldrich, USA</td><td style='width:153pt;'>P6744-1GA</td></tr><tr><td style='height:14.5pt;width:153pt;'>Na2HPO4-2H20</td><td style='width:164pt;'>Merck Millipore, Germany</td><td style='width:153pt;'>1065860500</td></tr><tr><td style='height:14.5pt;width:153pt;'>NaH2PO4-2H2O</td><td style='width:164pt;'>Merck Millipore, Germany</td><td style='width:153pt;'>1063451000</td></tr><tr><td style='height:14.5pt;width:153pt;'>NaCl</td><td style='width:164pt;'>Merck Millipore, Germany</td><td style='width:153pt;'>1064041000</td></tr><tr><td style='height:14.5pt;width:153pt;'>4N NaOH</td><td style='width:164pt;'>Carl Roth, Germany</td><td style='width:153pt;'>T198.1</td></tr><tr><td style='height:14.5pt;width:153pt;'>Thiopental</td><td style='width:164pt;'>Sandoz, Austria</td><td style='width:153pt;'>5,133</td></tr><tr><td style='height:14.5pt;width:153pt;'>Glycerol</td><td style='width:164pt;'>Sigma-Aldrich, USA</td><td style='width:153pt;'>G5516-500ML</td></tr><tr><td style='height:14.5pt;width:153pt;'>GenPure ultrapure water system</td><td style='width:164pt;'>Thermo Fisher Scientific, USA</td><td style='width:153pt;'>50131235</td></tr><tr><td style='height:14.5pt;width:153pt;'>Peristaltic pump</td><td style='width:164pt;'>ISMATEC, Germany</td><td style='width:153pt;'>ISM 930C</td></tr><tr><td style='height:14.5pt;width:153pt;'>Filter Paper</td><td style='width:164pt;'>MACHEREY-NAGEL, Germany</td><td style='width:153pt;'>MN 615 1/4</td></tr><tr><td style='height:14.5pt;width:153pt;'>Vibroslicer, VT1000S</td><td style='width:164pt;'>Leica Microsystems, Austria</td><td style='width:153pt;'>&#160;</td></tr><tr><td style='height:14.5pt;width:153pt;'>Ophthalmic scalpel</td><td style='width:164pt;'>Alcon Laboratories, USA</td><td style='width:153pt;'>&#160;</td></tr><tr><td style='height:14.5pt;width:153pt;'>Perfusion cannula</td><td style='width:164pt;'>Vieweg, Germany</td><td style='width:153pt;'>F560088-1</td></tr><tr><td style='height:14.5pt;width:470pt;' colspan='3'>High-pressure Freezing</td></tr><tr><td style='height:14.5pt;width:153pt;'>Copper carriers</td><td style='width:164pt;'>Engineering Office M. Wohlwend, CH</td><td style='width:153pt;'>528</td></tr><tr><td style='height:14.5pt;width:153pt;'>Sidol Polish</td><td style='width:164pt;'>Henkel, Germany</td><td style='width:153pt;'>&#160;</td></tr><tr><td style='height:15.75pt;width:153pt;'>Chamois skin</td><td style='width:164pt;'>Household supply store</td><td style='width:153pt;'>&#160;</td></tr><tr><td style='height:15.75pt;width:153pt;'>Hole punch, 1,5mm</td><td style='width:164pt;'>Stubai, Austria</td><td style='width:153pt;'>&#160;</td></tr><tr><td style='height:15.75pt;width:153pt;'>Denatured ethanol</td><td style='width:164pt;'>Donauchem, Austria</td><td style='width:153pt;'>&#160;</td></tr><tr><td style='height:15.75pt;width:153pt;'>Acetone</td><td style='width:164pt;'>Roth, Germany</td><td style='width:153pt;'>9372.5</td></tr><tr><td style='height:15.75pt;width:153pt;'>High Pressure Freezing Machine HPM 010</td><td style='width:164pt;'>BalTec, CH; now Leica Microsystems</td><td style='width:153pt;'>HPM010</td></tr><tr><td style='height:15.75pt;width:153pt;'>Stereo-microscope</td><td style='width:164pt;'>Olympus, Japan</td><td style='width:153pt;'>SZX10</td></tr><tr><td style='height:15.75pt;width:153pt;'>Liquid nitrogen</td><td style='width:164pt;'>&#160;</td><td style='width:153pt;'>&#160;</td></tr><tr><td style='height:15.75pt;width:153pt;'>Cryo-vials</td><td style='width:164pt;'>Roth, Germany</td><td style='width:153pt;'>E309.1</td></tr><tr><td style='height:15.75pt;width:153pt;'>CryoCane</td><td style='width:164pt;'>Nalge Nunc International,USA</td><td style='width:153pt;'>5015-0001</td></tr><tr><td style='height:15.75pt;width:153pt;'>CryoSleeve</td><td style='width:164pt;'>Nalge Nunc International,USA</td><td style='width:153pt;'>5016-0001</td></tr><tr><td style='height:15.75pt;width:153pt;'>Liquid nitrogen storage vessel</td><td style='width:164pt;'>Cryopal, France</td><td style='width:153pt;'>GT38</td></tr><tr><td style='height:15.75pt;width:153pt;'>Non-ionic detergent (Lavocid)</td><td style='width:164pt;'>Werner &amp; Mertz Professional, Germany</td><td style='width:153pt;'>&#160;</td></tr><tr><td style='height:15.75pt;width:470pt;' colspan='3'>Freeze-fracture and Replication</td></tr><tr><td style='height:15.75pt;width:153pt;'>Sandblaster, Mikromat 200-1</td><td style='width:164pt;'>JOKE Joisten &amp; Kettenbaum, Germany</td><td style='width:153pt;'>SANDURET 2-K</td></tr><tr><td style='height:15.75pt;width:153pt;'>Siliciumcarbid SIC 360, grain size 25 - 21&#181;</td><td style='width:164pt;'>JOKE Joisten &amp; Kettenbaum, Germany</td><td style='width:153pt;'>955932</td></tr><tr><td style='height:15.75pt;width:153pt;'>Freeze Fracture System BAF 060</td><td style='width:164pt;'>BalTec, CH; now Leica Microsystems</td><td style='width:153pt;'>BAF060</td></tr><tr><td style='height:15.75pt;width:153pt;'>Ceramic 12 well plate</td><td style='width:164pt;'>Gr&#246;pel, Austria</td><td style='width:153pt;'>14511</td></tr><tr><td style='height:15.75pt;width:153pt;'>Trizma base</td><td style='width:164pt;'>SIGMA, USA</td><td style='width:153pt;'>T1503</td></tr><tr><td style='height:15.75pt;width:153pt;'>Trizma hydrochloride</td><td style='width:164pt;'>SIGMA, USA</td><td style='width:153pt;'>T3253</td></tr><tr><td style='height:15.75pt;width:153pt;'>Sodium chloride</td><td style='width:164pt;'>Merck, Germany</td><td style='width:153pt;'>1,06,40,41,000</td></tr><tr><td style='height:15.75pt;width:153pt;'>SDS, Sodium lauryl sulfate</td><td style='width:164pt;'>Roth, Germany</td><td style='width:153pt;'>5136.1</td></tr><tr><td style='height:15.75pt;width:153pt;'>Sucrose</td><td style='width:164pt;'>Merck, Germany</td><td style='width:153pt;'>1,07,68,71,000</td></tr><tr><td style='height:15.75pt;width:153pt;'>TRIS</td><td style='width:164pt;'>Roth, Germany</td><td style='width:153pt;'>5429.3</td></tr><tr><td style='height:15.75pt;width:153pt;'>Universal Hybridization Oven</td><td style='width:164pt;'>Binder, Germany</td><td style='width:153pt;'>7001-0050</td></tr><tr><td style='height:15.75pt;width:470pt;' colspan='3'>Immunolabelling</td></tr><tr><td style='height:15.75pt;width:153pt;'>BSA</td><td style='width:164pt;'>SIGMA, USA</td><td style='width:153pt;'>A9647</td></tr><tr><td style='height:15.75pt;width:153pt;'>Anti-GFP Antibody</td><td style='width:164pt;'>Molecular Probes, USA</td><td style='width:153pt;'>A11122</td></tr><tr><td style='height:15.75pt;width:153pt;'>Anti-pan-AMPAR Antibody</td><td style='width:164pt;'>Frontier Institute, Japan</td><td style='width:153pt;'>pan AMPAR-GP-Af580-1</td></tr><tr><td style='height:15.75pt;width:153pt;'>Anti-NMDAR1 Antibody, clone 54.1</td><td style='width:164pt;'>Merck Millipore, Germany</td><td style='width:153pt;'>MAB363</td></tr><tr><td style='height:15.75pt;width:153pt;'>Opioid Receptor-Mu (MOR) Antibody</td><td style='width:164pt;'>ImmunoStar, USA</td><td style='width:153pt;'>24216</td></tr><tr><td style='height:15.75pt;width:153pt;'>EM goat anti-guinea pig, 5nm; secondary antibody</td><td style='width:164pt;'>BBInternational,</td><td style='width:153pt;'>EM.GAG5</td></tr><tr><td style='height:15.75pt;width:153pt;'>EM goat anti-rabbit, 15nm; secondary antibody</td><td style='width:164pt;'>BBInternational,</td><td style='width:153pt;'>EM.GAR15</td></tr><tr><td style='height:15.75pt;width:153pt;'>Donkey anti-rabbit, 10nm, secondary antibody</td><td style='width:164pt;'>AURION, Netherlands</td><td style='width:153pt;'>DAR 10nm</td></tr><tr><td style='height:15.75pt;width:153pt;'>Copper grids, 100 Parallel Bar</td><td style='width:164pt;'>Agar scientific, UK</td><td style='width:153pt;'>G2012C</td></tr><tr><td style='height:15.75pt;width:153pt;'>Incubator</td><td style='width:164pt;'>Major Science, USA</td><td style='width:153pt;'>MO-RC</td></tr><tr><td style='height:15.75pt;width:153pt;'>Pioloform Powder</td><td style='width:164pt;'>Agar scientific, UK</td><td style='width:153pt;'>R1275</td></tr><tr><td style='height:15.75pt;width:153pt;'>Chloroform</td><td style='width:164pt;'>Roth, Germany</td><td style='width:153pt;'>3313.1</td></tr></tbody></table></figure>

<a href='https://app.jove.com/v/53853/combined-optogenetic-freeze-fracture-replica-immunolabeling-to'>Source: Sch&#246;nherr, S., et. al. Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala. J. Vis. Exp. (2016)</a>This video demonstrates the immunolabelling of freeze-fractured replicas of mouse brain tissue for electron microscopy. The fractured and coated replicas are digested to expose receptors. Primary and gold-tagged secondary antibodies are added to label specific neuronal membrane receptors. The replica is mounted on a grid and visualized under an electron microscope.

<figure class='image image-style-align-center image_resized' style='display:table;margin-left:auto;margin-right:auto;width:25%;'><img style='aspect-ratio:1200/1333;' src='https://cloudfront.jove.com/files/ftp_upload/23313/23313_Figure_1_editor_23313.jpg' alt='23313_Figure_1.jpg' /></figure>Figure 1. Tissue Preparation and High-pressure Freezing. (A) Vibroslicer is used to section the tissue. (B) A resulting coronal mouse brain section containing the amygdala is shown side by side with a copp...

Source: Sch&#246;nherr, S., et. al. Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate ...

Take a freeze-fractured specimen of mouse brain tissue in a buffer.The specimen is coated with platinum to enhance surface details and with carbon for structural stability.Transfer the specimen to a vial with a digestion buffer containing detergent.&#160;Incubate to facilitate tissue breakdown, leaving intact the membrane and proteins embedded in the coating.Wash with buffer to remove debris.Add a blocking solution to prevent non-specific antibody binding.Introduce primary antibodies specific to target neuronal membrane protein.Wash with buffer to remove unbound antibodies.Incubate with gold-conjugated secondary antibodies that bind to primary antibodies.&#160;Wash with buffer to remove excess antibodies.Rinse with water to prevent artifacts from salt residues during microscopy.Mount the specimen on a transmission electron microscope or TEM grid.Under the TEM, visualize the target neuronal membrane protein identified by gold particles, which appear as black dots.

7 조회수. Immunolabeling of Neuronal Membrane Proteins in a Freeze-fractured Specimen of Mouse Brain Tissue

비디오: Immunolabeling of Neuronal Membrane Proteins in a Freeze-fractured Specimen of Mouse Brain Tissue - 실험

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

설치류 중추 신경계의 생체 Optogenetic 자극

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

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JoVE Journal

신경과학

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

효소 기반의 바이오 센서를 사용하면 알츠하이머 병 마우스 모델에서 토닉과 Phasic이 품목을 측정하는

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

고압 냉동, 전자 레인지를 이용한 콘트라스트 향상, 그리고 볼륨 이미지에 대 한 포함 하는 최소한의 수 지에 의해 생물학 견본 준비

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Immunolabeling of Neuronal Membrane Proteins in a Freeze-fractured Specimen of Mouse Brain Tissue

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Immunolabeling of Neuronal Membrane Proteins in a Freeze-fractured Specimen of Mouse Brain Tissue