Retrograde and Anterograde Tracing of Neural Projections

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The Journal of Neuroscience
Dec 2012



Neurons consist of four elements, the soma, dendrite, axon and terminal. They work in concert as the input (soma and dendrite) and output (axon and terminal) parts of neuronal transmission. To function and maintain neuronal activity and metabolisms, proteins and organelles should be transported from soma to terminal via anterograde axonal transport, and also from terminal to soma via retrograde transport. By utilizing these transport systems, neural projection is traced by injecting tracers into local sites of interest. Furthermore, neurochemical properties, such as glutamatergic and GABAergic, can be determined by combining retrograde and anterograde tracing with fluorescent in situ hybridization and immunofluorescence.

Keywords: In situ hybridization (原位杂交), Neuronal tracer (神经示踪), Immunohistochemistry (免疫组织化学)

Materials and Reagents

  1. Chloral hydrate
  2. Instant glue
  3. Biotinylated dextran amine (BDA) (3,000 molecular weight, 10% solution in PBS) (Life Technologies, InvitrogenTM, catalog number: D-7135 )
  4. Alexa Fluor 488-conjugated cholera toxin subunit b (Alexa488-CTb, 0.5% solution in saline) (Life Technologies, InvitrogenTM, catalog number: C-34775 )
  5. Distilled H2O (dH2O)
  6. Pentobarbital
  7. Sodium azide
  8. Phosphate-buffered saline (PBS)
  9. Alexa Flour 594-conjugated streptavidin (Life Technologies, InvitrogenTM, catalog number: S-11227 )
  10. Normal donkey serum (10% solution in PBS) (Jackson Immuno Research, catalog number: 017-000-121 )
  11. Primary antibodies for immunofluorescence
    Note: We almost use self-made antibodies (Kudo et al., 2012).
  12. Secondary antibodies
    Note: We use products from Invitrogen and/or Jackson Immuno Research, whose host are donkey.
  13. Acetic anhydrate
  14. Triethanolamine-HCl
  15. Formadime
  16. Tris(hydroxymethyl) aminomethane (TRIS)
  17. Ficoll
  18. Polyvinylpyrrolidone
  19. Bovine serum albumin (BSA)
  20. tRNA
  21. Ethylenediaminetetraacetic acid (EDTA)
  22. N-Lauroylsarcosine sodium salt (NLS)
  23. Dextran sulfate
  24. cRNA probes (Kudo et al., 2012; Yamasaki et al., 2010)
  25. Sodium citrate
  26. Lodoamide
  27. DIG blocking reagent (Roche Applied Science, catalog number: 11096176001 )
  28. Normal sheep serum (Chemicon, catalog number: S22-100ML )
  29. Maleic acid
  30. Tyramide signal amplification (TSA) blocking reagent (PerkinElmer Life and Analytical Science, catalog number: FP1020 )
  31. Peroxidase-conjugated anti-DIG antibody (1:1,000 in DIG blocking buffer) (Roche Diagnostics, catalog number: 11207733910 )
  32. Cyanine 3 (Cy3) Amplification Reagent (PerkinElmer Life and Analytical Science, catalog number: FP1170 )
  33. 1x Plus Amplification Diluent (PerkinElmer Life and Analytical Science, catalog number: FP1135 )
  34. H2O2
  35. Anti-Alexa Fluor 488 antibody for detection of Alexa488-CTb (raised in rabbit) (1:1,000 dilution) (Invitrogen, catalog number: A-11094 )
  36. TOTO-3 iodide (642/660) (1:50 in PBS) (Life Technologies, InvitrogenTM, catalog number: T3604 )
  37. Primary antibody solution (1 μg/ml in PBS-T)
  38. Fluorophore-linked secondary antibody solution (1:200 in PBS-T)
  39. Digoxigenin (DIG)-labeled cRNA probes
  40. Dimethyl sulfoxide (DMSO)
  41. Blocking reagent (Roche Applied Science, catalog number: 11096176001)
  42. Saline (see Recipes)
  43. 3.5% chloral hydrate (see Recipes)
  44. 0.1 M PB (see Recipes)
  45. 5 N NaOH (see Recipes)
  46. 4% Paraformaldehyde (PFA) (see Recipes)
  47. 20% Tween-20 (see Recipes)
  48. PBS containing 0.1% Tween-20 (PBS-T) (see Recipes)
  49. 0.25% acetic anhydrate in 0.1 M triethanolamine-HCl (pH 8.0) (see Recipes)
  50. 1 M Tris-HCl (pH 7.4 or 8.0) (see Recipes)
  51. 5 M NaCl (see Recipes)
  52. 0.5 M ethylenediaminetetraacetic acid (EDTA) (see Recipes)
  53. Hybridization buffer (see Recipes)
  54. Standard saline citrate (SSC) (see Recipes)
  55. NaCl-Tris-EDTA (NTE) buffer (see Recipes)
  56. Tris-NaCl-Tween (TNT) buffer (see Recipes)
  57. 20 mM iodoamide in NTE buffer (see Recipes)
  58. DIG blocking solution (see Recipes)
  59. 0.5% TSA blocking buffer (see Recipes)
  60. Cy3-TSA amplification solution (see Recipes)


  1. Scissors
  2. Surgical knife
    Note: We use set of stainless-steel mess handle (Feather, No.3) and stainless-steel spare blade (Feather, No.14).
  3. Syringe
  4. Syringe needle
  5. Stereotaxic instrument (Narishige, model: SR-5M )
  6. Surgical cotton
  7. Pneumatic pump (Pneumatic Picopump) (World Precision Instruments, model: PV800 )
  8. Puller (Narishige, model: PC-10 )
  9. Glass pipette (Narishige, model: G-1.2 )
  10. Peristaltic pump (ATTO Corporation, model: SJ-1211H )
  11. Cork board
  12. Microslicer (Leica Microsystems, model: VT1000S )
  13. Razor blade
  14. 10 ml tube
  15. Confocal laser-scanning microscope (Olympus, model: FV1000 )
  16. Hybridization oven (Bellco, model: 7930-00110 )
  17. Water bath
  18. Fluorescence microscope
    Note: Fluorescence signals of Alexa Fluor 488 and Cy3 or Alexa Fluor 594 are observed through fluorescence mirror units (Olympus, model: U-MWIBA3 , U-MWIG3 ).
  19. Aspirator


  1. Confocal software (Olympus, FV10-ASW, ver.1.7)


  1. Injection of anterograde or retrograde tracer
    1. Prepare the tracer solution and all equipment needed (scissors, surgical knife, syringe needles etc).
      Note: BDA and Alexa488-CTb are used as an anterograde or retrograde tracer, respectively. Spin down tracer solutions before use.
    2. Anesthetize an animal with 3.5% chloral hydrate (350 mg/kg body weight, i.p.).
    3. Check the animal being anesthetized by loss of righting reflex and lack of response to hitching its paws.
    4. Place the anesthetized animal on stereotaxic instrument with locking the nose and ears.
      Note: First, insert the auxiliary ear bar into the mouse ear canal and fix it tightly, but be careful not to insert it too deeply to injure the inner ear. Next, align the vertical level of the nose clamp and hook the mouse tooth on it, and then hold down the nose. Check whether the mouse head do not move wobbly to finish fixation.
    5. Shave the hair and incise the scalp along the rostro-caudal axis.
    6. Clip the incised scalp using syringe needles bent into a hook and remove lamina.
      Note: To remove lamina, snick it by scissors and wipe the surface of the skull bone by surgical cottons.
    7. Using a surgical knife cut the skull bone and make a square hole at the position of tracer injection.
    8. Prepare a glass pipette using a puller.
    9. Fill a glass pipette with ~1 μl of the tracer solution by a capillary phenomenon and attach it to a tube connected with a pneumatic pump.
      Note: If the tip of the pipette is too fine to draw the tracer solution by a capillary phenomenon, snap the tip slightly. Moreover, too fine tip often prevents smooth injection as tiny concomitants get jammed in the pipette.
    10. Position the tip of the glass pipette on the bregma and then stereotaxically insert the pipette into the target region.
      Note: To make injection space, insert the pipette 0.1 mm deeper and turn back to the target region.
    11. Inject the tracer solution by air pressure at 10 psi with 5 sec intervals for 1 min.
    12. Leave the pipette inserted for 15 min, and then pull out it carefully.
    13. Replace the removed skull bone on the square hole and suture the scalp.
      Note: Instant glue is used for closing incision site as substitute for a surgical suture.
    14. Release and place the animal back into a home cage.
    15. Keep the animal for several days until fixation.
      Note: Determine the period of survival depending on the distance of projection.
      In our experiments (between the bed nucleus of the stria terminalis (BST) and the ventral tegmental area (VTA)), BDA-injected and Alexa488-CTb-injected animals are incubated for at least 4 and 2 days, respectively (Kudo et al., 2012).

  2. Perfusion and section preparation
    1. Place a beaker containing 4% PFA fixative solution (100 ml for each mouse) in ice bath. Set on peristaltic pump a silicone tube with one end put into the fixative beaker and the other end equipped with a 25 G syringe needle.
    2. Prepare all equipment needed (scissors, forceps, syringe needles to impale hands/feet etc).
    3. Run the peristaltic pump and fill up tubes and the syringe needle with the fixative solution.
    4. Deeply anesthetize the animal with overdosed pentobarbital (100 mg/kg of body weight, i.p.).
    5. After confirming the animal asleep, impale the animal’s hands and feet on a cork board with syringe needles.
    6. Open the abdominal cavity by horizontal cutting of the belly skin and muscles. Then cut the skin of the chest along the midline up to the jaw, and detach the skin from the chest wall.
    7. Cut the diaphragm to open the thoracic cavity, and then cut both sides of the chest wall to expose the heart. It is important not to injure the internal thoracic artery, which runs vertically along the sternum.
    8. Snick the wall of the right auricle by sharp scissors or the pit of syringe needle, and prick the left ventricle with a syringe needle connecting to the fixative beaker.
    9. Start transcardial perfusion for 10 min, so that 3 fixative volumes of the body weight run in 10 min.
    10. Excise a fixed brain and post-fix it for 2 h. Using a razor blade, divide the brain into two blocks with one containing a tracer-injected site and another containing neural regions of interest.
      Note: In our experiments, we divided the brain into two parts containing the BST and VTA, respectively, by coronal cutting between the hypothalamus and mammillary body (Kudo et al., 2012).
    11. Prepare sections of the fixed brain (50 μm in thickness) using a microslicer, whose buffer bath is filled with 0.1 M PB.
      Note: Especially in free-floating in situ hybridization experiment, the fixed brain block and sections should be kept in PB. When PBS is used for buffer, detection sensitivity of mRNA signal tends to be lowered.
    12. Collect sections in 24-well plate and store in 0.1 M PB containing 0.1% sodium azide.

  3. Anterograde tracing combined with fluorescence immunohistochemistry
    Note: This method is used to determine neurochemical properties of axon terminals, which are projected from anterograde tracer-injected regions. Vesicular glutamate transporters (VGluTs) and vesicular inhibitory transporter (VIAAT) are frequently used as excitatory (glutamatergic) and inhibitory (GABAergic and glycinergic) terminal markers, respectively (Kudo et al., 2012; Figure 1 in this manual). By using other neurochemical markers for combination with anterograde tracing, one can also determine whether the projection is cholinergic, serotonergic, dopaminergic, adrenergic, histeminergic, or peptidergic.
    1. Prepare sections from BDA-injected brains.
    2. Select sections containing the injected region and visualize BDA signals by incubation in Alexa Flour 594-conjugated streptavidin (1:500 in PBS) for 10 min. Photograph the injection site of BDA using a fluorescence microscope.
    3. Submerge 1~3 sections for neurochemical testing in a 10 ml tube containing PBS containing 0.1% Tween-20 (PBS-T) for 10 min.
    4. Block with 10% normal donkey serum in PBS for 20 min.
      Note: Blocking and antibody solutions are ~0.5 ml per tube, and washing solutions are ~10 ml per tube in each wash. Solution exchange is efficient by using aspirator, but should be carefully done not to aspirate or dry up sections.
    5. Incubate in primary antibody solution (1 μg/ml in PBS-T) overnight.
    6. Wash with PBS-T for 5 min three times.
    7. Incubate in fluorophore-linked secondary antibody solution (1:200 in PBS-T) for 2 h for neurochemical marker detection.
    8. Wash with PBS-T for 5 min three times.
    9. Wash with PBS briefly.
    10. Incubate in Alexa Flour 594-conjugated streptavidin (1:500 in PBS) for 10 min for BSA detection.
    11. Wash with PBS for 5 min three times.
    12. Mount sections on glass slides, make coverslip, and observe using a fluorescence microscope.
      Note: Images are captured using a confocal laser scanning microscope, digitized at 12 bit resolution into an array of 640 x 640 pixels (pixel size, 0.1 μm). To investigate the neurochemical characteristic of BDA-labeled axon terminals, take images (x60 magnifications, x3 zoom) of the traced region from each slice. For analysis, we counted terminal marker-positive boutons whose center point matched with that of BDA labeling. Bouton-like structures which are only labeled with BDA are often observed. If they are not labeled with other terminal markers, they should not be used for counting (Figure 1).

      Figure 1. Combined anterograde tracer labeling and immunofluorescence. BDA is injected into the bed nucleus of the stria terminalis (BST) and anterogradely transported to the ventral tegmental area (VTA). In sections containing the VTA, BDA staining (red) and immunofluorescence for type 2 vesicular glutamate transporter (VGluT2; green) and vesicular inhibitory amino acid transporter (VIAAT; blue) are performed. Images are used for counting (x60 magnifications, x3 zoom). VGluT2-positive BDA axon terminals (arrows) and VIAAT-positive BDA axon terminals (arrowheads) are identified as glutamatergic and GABAergic afferents from the BST, respectively. Scale bars: 10 μm.

  4. Retrograde tracing combined with free-floating fluorescent in situ hybridization (FISH)
    Note: This method is used to determine neurochemical properties of neurons, which project their axons to retrograde tracer-injected regions. It is recommended to prepare sections and start FISH incubation on the same day, because detection sensitivity is drastically decreased after sectioning. Expression of VGluTs and glutamic acid decarboxylase (GAD) (or VIAAT) mRNAs are used to determine the neurochemical properties of excitatory and inhibitory neurons, respectively (Kudo et al., 2012; Figure 2 in this paper).
    1. Prepare sections from Alexa488-CTb-injected brains.
    2. Check the injection site of Alexa488-CTb using a fluorescence microscope.
    3. Submerge a single section in a 10 ml tube containing PB.
      Note: It is recommended that one tube is used for one section, because more than two sections per tube decrease reaction sensitivity. For free-floating FISH, solutions for hybridization, antibody reaction, inactivation of peroxidases, fluorescence detection and counterstaining are ~0.5 ml per tube, and washing solutions are ~10 ml per tube in each wash. Solution exchange is efficient by using aspirator, but should be carefully done not to aspirate or dry up sections.
    4. Acetylate sections with 0.25% acetic anhydrate in 0.1 M triethanolamine-HCl (pH 8.0) for 10 min.
      Note: Prepare the solution at time of use.
    5. Prehybridize in hybridization buffer for 1 h.
    6. Hybridize in hybridization buffer supplemented with digoxigenin (DIG)-labeled cRNA probes at a dilution of 1:1,000, performed at 63.5 °C for 12 h.
      Note: Preparation of DIG-labeled cRNA probes is described in previous our reports (Kudo et al., 2012; Yamasaki et al., 2010). Hybridization is performed in a hybridization oven. Cover the tube tip with parafilm to avoid drying off.
    7. Wash with 5x SSC for 30 min, 4x SSC containing 50% formamide (Formamide 1) for 40 min, 2x SSC containing 50% formamide (Formamide 2) for 40 min and 0.1x SSC for 15 min, performed at 61 °C.
      Note: Solutions are prewarmed at 61 °C in hot bath, and washing steps are performed in hot bath.
    8. Incubate in 0.1x SSC for 15 min, NTE buffer for 20 min, 20 mM iodoamide in NTE buffer for 20 min, NTE buffer for 10 min and TNT buffer for 10 min, performed at room temperature (RT).
      Note: All subsequent steps are performed at RT.
    9. Block sections with DIG blocking buffer for 30 min and 0.5% TSA blocking buffer for 30 min.
    10. Incubate in peroxidase-conjugated anti-DIG antibody in DIG blocking buffer (1:1,000) for 2 h.
    11. Wash with TNT buffer for 15 min twice.
    12. Detect signals for peroxidase using Cy3-TSA plus amplification kit system (see Recipe 20) for 10 min, performed in shade.
    13. Wash with TNT buffer for 5 min three times.
    14. For inactivation of residual peroxidase activities, incubate in 3% H2O2 in TNT buffer for 30 min.
      Note: This inactivation step is important to enhance contrast of signals. If this step is skipped, background signals remain strong as well as true-positive signals.
    15. Wash with TNT buffer for 5 min three times.
    16. Check the fluorescence signals for target mRNAs using a fluorescence microscope.
      Note: Because the fluorescence of Alexa488-CTb becomes weak and readily extincts after hybridization and the post-hybridization wash, the tracer should be detected by immunofluorescence using anti-Alexa Fluor 488 antibody, as shown in Procedure III.
    17. After immunofluorescence for Alexa488-CTb, counterstain with TOTO-3 (1:50 in PBS) for 20 min.
    18. Mount sections on glass slides and observe using a fluorescence microscope.
      Note: To investigate the neurochemical composition of retrogradely traced neurons, collect tiled images to cover the whole traced region (x20 magnifications, x1.3 zoom), and counted the number of positive cells having the nucleus (show TOTO-3 signal) (Figure 2).

      Figure 2. Combined retrograde tracer labeling and fluorescence in situ hybridization. Alexa488-CTb is injected into the VTA and retrogradely transported to the BST. In sections containing the BST, immunofluorescence for Alexa Fluor 488 (green), fluorescence in situ hybridization for GAD mRNA (red) and counterstaining by TOTO-3 (blue) are performed. The top image is a part of tiled image used for counting (x20 magnifications, x1.3 zoom) and bottom images are higher magnification (x60 magnifications, x1.0 zoom). Neurons co-labeled for GAD mRNA and CTb (arrows) are identified as VTA-projecting GABAergic neurons. Scale bar: top, 100 μm; bottom, 30 μm.


  1. Saline
    NaCl 9 g/L of dH2O
  2. 3.5% chloral hydrate
    Chloral hydrate 1.75 g/50 ml of dH2O
  3. 0.1 M PB
    To make 1 L of 0.1 M PB
    Mix 2.95 g of NaH2PO4.2H2O and 29 g of Na2HPO4.12H2O
    Add dH2O to 1 L and stir ~ 30 min
    Store at 4 °C
  4. 5 N NaOH
    NaOH 40 g/200 ml of dH2O
  5. 4% PFA
    To make 1 L of PFA
    Make 500 ml of 8% PFA (Solution (a))
    Heat 500 ml dH2O to 80 °C (Do NOT boil)
    Add 40 g of PFA powder and stir ~10 min
    Add 250 μl of 5 N NaOH and keep stirring until the solution gets clear
    Next, make 500 ml of 0.2 M PB (Solution (b)) in another beaker
    Mix 2.95 g of NaH2PO4.2H2O and 29 g of Na2HPO4.12H2O
    Add dH2O to 500 ml and stir ~ 30 min
    Mix solution (a) and solution (b) and stir ~5 min
    Filtrate and store at 4 °C
  6. PBS
    To make 1 L of 10x PBS stock solution
    Mix 87 g of NaCl, 3.1 g of NaH2PO4.2H2O and 28.7 g of Na2HPO4.12H2O
    Add dH2O to1 L and stir ~ 2 h
    Store at RT
    Dilute this 10x stock solution by 1/10 using dH2O
  7. 20% Tween-20
    Mix 20 ml of Tween-20 with 80 ml of ddH2O by stirring
    Store at 4 °C
  8. PBS containing 0.1% Tween-20 (PBS-T)
    Add 2.5 ml of 20% Tween-20 with 500 ml of PBS
    Store at RT
  9. 0.25% acetic anhydrate in 0.1 M triethanolamine-HCl (pH 8.0)
    To make 100 ml of acetylation solution
    Mix triethanolamine-HCl with 100 ml of dH2O by stirring
    Add 950 μl of 5 N NaOH and make sure that pH is 8.0
    Add 250 μl of acetic anhydride (*add just before use) and stir ~3 min
  10. 1 M Tris-HCl (pH 7.4 or 8.0)
    To make 1 L of Tris-HCl buffer
    Mix 121.1 g of Tris base (tris(hydroxymethyl)aminomethane) with 800 ml of dH2O
    pH to 7.4 or 8.0 with HCl
    Add dH2O to 1 L
    Autoclave and then store at RT
  11. 5 M NaCl
    To make 1 L of 5 M NaCl
    Mix 292.2 g of NaCl with 800 ml of dH2O by stirring
    Add dH2O to 1 L and stir until they dissolve completely
    Autoclave and then store at RT
  12. 0.5 M ethylenediaminetetraacetic acid (EDTA)
    To make 500 ml of 0.5 M EDTA
    Mix 93.1 g of EDTA with 400 ml of dH2O
    pH to 8.0 with 5 N NaOH
    Add dH2O to 500 ml
    Autoclave and then store at RT
  13. Hybridization buffer
    To make 500 ml of hybridization buffer
    Autoclave two 500 ml beakers; one is filled with 250 ml of dH2O and another is empty
    After beakers cool down, pour 80 ml of autoclaved dH2O and 250 ml of formamide into the empty beaker
    Add reagents described on the following table
    Reagent Quantity
    1 M Tris-HCl (pH 8.0)
    16.5 ml
    100 mg
    100x Denhardt's*
    5 ml
    5M NaCl
    60 ml
    0.5 M EDTA
    1 ml
    0.5 g
    Dextran sulfate
    50 g
    *100x Denhardt's
    Quantity (for 50 ml)
    Final concentration (100x)
    1 g
    2% (w/v)
    1 g
    2% (w/v)
    1 g
    2% (w/v)
    to 50 ml
    Add autoclaved dH2O to 500 ml and then dissolve on shaking for ~24 h.
    Filtrate and then store at -30 °C.
  14. Standard saline citrate (SSC)
    To make 1 L of 20x SSC stock solution
    Mix 175.3 g of NaCl and 88.2 g of sodium citrate
    Add dH2O to1 L and stir ~ 2 h
    Autoclaved and then store at RT
    Dilute this 20x stock solution as described on the following table

    5x SSC
    (for 1 L)
    0.1x SSC
    (for 1 L)
    Formamide 1
    (for 150 ml)
    Formamide 2
    (for 150 ml)
    20x SSC
    250 ml
    5 ml
    30 ml
    15 ml
    20% Tween-20
    25 μl
    25 μl
    7.5 μl
    7.5 μl
    75 ml
    75 ml
     to 1 L
    to 1 L
    to 150 ml
    to 150 ml
    Store at RT
  15. NaCl-Tris-EDTA (NTE) buffer
    To make 1 L of NTE buffer
    Mix 100 ml of 5 M NaCl, 10 ml of 1 M Tris-HCl (pH 8.0), 10 ml of 0.5 M EDTA and 25 μl of 20% Tween-20
    Add dH2O to1 L and store at RT
  16. Tris-NaCl-Tween (TNT) buffer
    To make 1 L of TNT buffer
    Mix 30 ml of 5 M NaCl, 100 ml of 1 M Tris-HCl (pH 7.4) and 25 μl of 20% Tween-20
    Add dH2O to1 L and store at RT
  17. 20 mM iodoamide in NTE buffer
    Iodoacetamide 0.37 g/100 ml of NTE buffer
  18. DIG blocking solution
    Normal sheep serum: 10% blocking reagent*: TNT buffer = 1:1:8
    *10% blocking reagent (for 10 ml)
    1. Prepare maleic acid buffer
      To make 500 ml of maleic acid buffer
      Mix 58.1 g of maleic acid and 43.9 g of NaCl with 400 ml of dH2O
      pH to 7.5 with 5 N NaOH
    2. Dissolve 1 g of blocking reagent to 10 ml with the maleic acid buffer with shaking and heating
    3. Autoclave and then store at -80 °C
  19. 0.5% TSA blocking solution
    Add 0.5 g of blocking reagent to 100 ml of TNT buffer
    To dissolve the blocking reagent, heat to 60 °C for 1 h with stirring
    Store at -20 °C
  20. Cy3-TSA amplification solution
    Add 60 μl of DMSO to Cy3 Amplification Reagent (Cy3 solution; store at 4 °C)
    Dilute this Cy3 solution in 1x Plus Amplification Diluent (1:200)


This protocol is adapted from Kudo et al. (2012).


  1. Kudo, T., Uchigashima, M., Miyazaki, T., Konno, K., Yamasaki, M., Yanagawa, Y., Minami, M. and Watanabe, M. (2012). Three types of neurochemical projection from the bed nucleus of the stria terminalis to the ventral tegmental area in adult mice. J Neurosci 32(50): 18035-18046.
  2. Yamasaki, M., Matsui, M. and Watanabe, M. (2010). Preferential localization of muscarinic M1 receptor on dendritic shaft and spine of cortical pyramidal cells and its anatomical evidence for volume transmission. J Neurosci 30(12): 4408-4418.


神经元由四个元素组成,即索马,树突,轴突和末端。 它们作为神经元传播的输入(神经和树突)和输出(轴突和末端)部分协同工作。 为了起到和维持神经元活动和代谢的作用,蛋白质和细胞器应通过逆行轴突运输从脊髓运输到终末,也可以通过逆行运输从末端转移到血管。 通过利用这些运输系统,通过将示踪剂注入到感兴趣的地方来跟踪神经投影。 此外,神经化学性质,如谷氨酸能和GABA能,可以通过逆行和顺行跟踪与荧光原位杂交和免疫荧光结合来确定。

关键字:原位杂交, 神经示踪, 免疫组织化学


  1. 水合氯化
  2. 速溶胶
  3. 将生物素化的葡聚糖胺(BDA)(3,000分子量,10%PBS溶液)(Life Technologies,Invitrogen TM,目录号:D-7135)
  4. Alexa Fluor 488-缀合的霍乱毒素亚基b(Alexa488-CTb,0.5%的盐水溶液)(Life Technologies,Invitrogen TM,目录号:C-34775)
  5. 蒸馏H 2 O(dH 2 O)
  6. 戊巴比妥
  7. 叠氮化钠
  8. 磷酸盐缓冲盐水(PBS)
  9. Alexa Flour 594-缀合的链霉亲和素(Life Technologies,Invitrogen TM,目录号:S-11227)
  10. 正常驴血清(10%PBS溶液)(Jackson Immuno Research,目录号:017-000-121)
  11. 免疫荧光的主要抗体
    注意:我们几乎使用自制抗体(Kudo et al。,2012)。
  12. 二抗
    注意:我们使用来自Invitrogen和/或Jackson Immuno Research的产品,其主机是驴。
  13. 乙酸酐
  14. 三乙醇胺 - HCl
  15. Formadime
  16. 三(羟甲基)氨基甲烷(TRIS)
  17. Ficoll
  18. 聚乙烯吡咯烷酮
  19. 牛血清白蛋白(BSA)
  20. tRNA
  21. 乙二胺四乙酸(EDTA)
  22. N-月桂酰肌氨酸钠盐(NLS)
  23. 硫酸葡聚糖
  24. cRNA探针(Kudo等人,2012; Yamasaki等人,2010)
  25. 柠檬酸钠
  26. 洛洛酰胺
  27. DIG封闭试剂(Roche Applied Science,目录号:11096176001)
  28. 正常绵羊血清(Chemicon,目录号:S22-100ML)
  29. 马来酸
  30. 酪胺信号放大(TSA)阻断试剂(PerkinElmer Life and Analytical Science,目录号:FP1020)
  31. 过氧化物酶缀合的抗DIG抗体(在DIG封闭缓冲液中1:1000)(Roche Diagnostics,目录号:11207733910)
  32. 花青3(Cy3)扩增试剂(PerkinElmer Life and Analytical Science,目录号:FP1170)
  33. 1x Plus扩增稀释剂(PerkinElmer Life and Analytical Science,目录号:FP1135)
  34. H 2 2
  35. 用于检测Alexa488-CTb(在兔中生长)(1:1,000稀释)(Invitrogen,目录号:A-11094)的抗Alexa Fluor 488抗体
  36. TOTO-3碘化物(642/660)(PBS中1:50)(Life Technologies,Invitrogen TM,目录号:T3604)
  37. 第一抗体溶液(PBS-T中1μg/ml)
  38. 荧光团连接的二抗溶液(PBS-T中1:200)
  39. 地高辛(DIG)标记的cRNA探针
  40. 二甲基亚砜(DMSO)
  41. 封闭试剂(Roche Applied Science,目录号:11096176001)
  42. 盐水(见配方)
  43. 3.5%水合氯醛(见配方)
  44. 0.1 M PB(参见配方)
  45. 5 N NaOH(见配方)
  46. 4%多聚甲醛(PFA)(参见配方)
  47. 20%Tween-20(见配方)
  48. 含有0.1%Tween-20(PBS-T)的PBS(参见Recipes)
  49. 0.25%无水乙酸的0.1M三乙醇胺-HCl(pH8.0)(参见配方)
  50. 1 M Tris-HCl(pH 7.4或8.0)(参见配方)
  51. 5 M NaCl(见配方)
  52. 0.5 M乙二胺四乙酸(EDTA)(见配方)
  53. 杂交缓冲液(参见配方)
  54. 标准盐水柠檬酸盐(SSC)(见配方)
  55. NaCl-Tris-EDTA(NTE)缓冲液(参见配方)
  56. Tris-NaCl-Tween(TNT)缓冲液(见配方)
  57. 20mM碘酰胺的NTE缓冲液(参见配方)
  58. DIG封锁解决方案(参见配方)
  59. 0.5%TSA封闭缓冲液(见配方)
  60. Cy3-TSA放大溶液(参见配方)


  1. 剪刀
  2. 手术刀
  3. 注射器
  4. 注射针
  5. 立体定位仪(Narishige,型号:SR-5M)
  6. 手术棉
  7. 气动泵(气动Picopump)(世界精密仪器,型号:PV800)
  8. Puller(Narishige,型号:PC-10)
  9. 玻璃移液管(Narishige,型号:G-1.2)
  10. 蠕动泵(ATTO公司,型号:SJ-1211H)
  11. 软木板
  12. 微型切片机(Leica Microsystems,型号:VT1000S)
  13. 剃刀刀片
  14. 10ml管
  15. 共聚焦激光扫描显微镜(Olympus,型号:FV1000)
  16. 杂交炉(Bellco,型号:7930-00110)
  17. 水浴
  18. 荧光显微镜
    注意:通过荧光镜单元(Olympus,型号:U-MWIBA3,U-MWIG3)观察Alexa Fluor 488和Cy3或Alexa Fluor 594的荧光信号。
  19. 吸气器


  1. 共聚焦软件(Olympus,FV10-ASW,ver.1.7)


  1. 注射顺行或逆行示踪剂
    1. 准备示踪剂溶液和所有需要的设备(剪刀,手术刀,注射器针等)。
      注意:BDA和Alexa488-CTb分别用作顺行或逆行示踪剂。在使用之前先旋转 下来的示踪剂解决方案。
    2. 麻醉动物与3.5%水合氯醛(350毫克/公斤体重,i.p.)
    3. 检查动物被正确的反射失去麻醉和缺乏对爪子的反应。
    4. 将麻醉的动物放在立体定位仪上,锁定鼻子和耳朵 注意:首先,将辅助耳杆插入鼠标耳道并将其固定,但要小心不要将其过深插入以伤害内耳。接下来,对准鼻夹的垂直高度,并将鼠标齿挂在它上面,然后按住鼻子。检查鼠标头是否不摇晃,以完成固定。
    5. 剃去头发,沿着罗斯特 - 尾部轴切开头皮。
    6. 使用注射器针头夹入切开的头皮,将其弯成钩子并移除层板 注意:要移除椎板,用剪刀剪刀,并用手术棉布擦拭颅骨表面。
    7. 使用手术刀切开颅骨,并在示踪剂注射的位置形成一个方形孔
    8. 使用拉片准备玻璃吸管。
    9. 通过毛细管现象向玻璃吸管中加入约1μl示踪剂溶液,并将其连接到与气动泵连接的管子。
    10. 将玻璃吸管的尖端放置在前囟上,然后立体定位地将移液管插入目标区域。
    11. 通过空气压力以10psi注入示踪剂溶液,间隔5秒,持续1分钟
    12. 让移液器插入15分钟,然后小心地拉出。
    13. 将取出的颅骨置于方孔上,缝上头皮。
    14. 释放并将动物放回笼子。
    15. 保持动物数天,直到固定。
      在我们的实验中(在纹状体末端的床核(BST)和腹侧被盖区(VTA)之间),将BDA注射的和Alexa488-CTb注射的动物分别孵育至少4天和2天 Kudo et al。,2012)。

  2. 灌注和切片准备
    1. 将含有4%PFA固定液(每只小鼠100ml)的烧杯置于冰浴中。 将蠕动泵置于蠕动泵上,将硅胶管的一端放入固定烧杯中,另一端安装25G注射器针。
    2. 准备所需的所有设备(剪刀,镊子,注射器针头刺穿手/脚等)。
    3. 运行蠕动泵,并用固定溶液填充管和注射器针
    4. 用过量的戊巴比妥(100mg/kg体重,i.p。)深度麻醉动物。
    5. 在确认动物睡着后,用注射器针刺入动物的手和脚在软木板上。
    6. 通过水平切割腹部皮肤和肌肉打开腹腔。然后沿着中线切开胸部皮肤,直到下巴,并从胸壁分离皮肤
    7. 切开隔膜打开胸腔,然后切开胸壁的两侧以暴露心脏。重要的是不要伤害沿胸骨垂直延伸的胸内动脉。
    8. 用锋利的剪刀或注射器针头的坑戳击右耳廓的壁,用连接到固定烧杯的注射器针刺入左心室。
    9. 开始心脏灌注10分钟,以便3个固定体积的体重在10分钟内运行
    10. 消除固定的大脑和后固定它2小时。使用剃刀刀片,将大脑分成两个块,一个包含示踪剂注射部位,另一个包含感兴趣的神经区域。
    11. 使用微量切片机制备固定脑(厚度为50μm)的切片,其缓冲液用0.1M PB填充。
    12. 收集24孔板中的切片并存储在含有0.1%叠氮化钠的0.1M PB中
  3. 顺行跟踪结合荧光免疫组化
    1. 从BDA注射的大脑准备切片。
    2. 选择含有注射区域的切片并通过在Alexa Flour 594缀合的链霉亲和素(在PBS中1:500)孵育10分钟来显现BDA信号。 使用荧光显微镜照片BDA的注射部位。
    3. 在含有含有0.1%Tween-20(PBS-T)的PBS的10ml管中浸没1〜3个神经化学测试切片10分钟。
    4. 用10%正常驴血清在PBS中封闭20分钟 注意:阻断和抗体溶液每管约0.5ml,每次洗涤的洗涤溶液为〜10ml。 通过使用吸气器,溶液交换是高效的,但应小心不要吸入或干燥部分。
    5. 在一抗溶液(1μg/ml,在PBS-T中)孵育过夜
    6. 用PBS-T洗涤5分钟三次。
    7. 在荧光团连接的二抗溶液(PBS-T中1:200)孵育2小时以进行神经化学标记物检测。
    8. 用PBS-T洗涤5分钟三次。
    9. 短暂用PBS清洗。
    10. 孵育在Alexa面粉594共轭链霉亲和素(PBS中1:500)10分钟进行BSA检测。
    11. 用PBS洗涤5分钟三次。
    12. 在玻片上安装切片,制成盖玻片,并使用荧光显微镜观察 注意:使用共聚焦激光扫描显微镜捕获图像,以12位分辨率数字化为640×640像素(像素尺寸,0.1μm)的阵列。为了研究BDA标记的轴突末端的神经化学特性,从每个切片拍摄描绘区域的图像(x60放大率,x3放大)。为了分析,我们计数终点标记阳性boutons的中心点与BDA标记的匹配。通常观察到仅用BDA标记的bouton样结构。如果它们没有标记其他终端标记,它们不应该用于计数(图1)。

      图1.组合的顺行示踪剂标记和免疫荧光。 BDA注射到纹状体末端(BST)的床核中,并顺行转运到腹侧被盖区(VTA)。在含有VTA的切片中,进行2型囊泡谷氨酸转运蛋白(VGluT2;绿色)和囊泡抑制性氨基酸转运蛋白(VIAAT;蓝色)的BDA染色(红色)和免疫荧光。图像用于计数(x60放大率,x3缩放)。 VGluT2-阳性BDA轴突末端(箭头)和VIAAT-阳性BDA轴突末端(箭头)分别被鉴定为来自BST的谷氨酸能和GABA能神经传入。比例尺:10μm
  4. 逆行跟踪与自由浮动荧光原位杂交(FISH)结合
    注意:这种方法用于确定神经元的神经化学性质,其将它们的轴突投影到逆行示踪剂注射区域。建议准备切片并在同一天开始FISH孵育,因为检测灵敏度在切片后显着降低。 VGluTs和谷氨酸脱羧酶(GAD)(或VIAAT)mRNA的表达分别用于确定兴奋性和抑制性神经元的神经化学性质(Kudo等,2012;本文中的图2)。
    1. 从注射Alexa488-CTb的大脑准备切片。
    2. 使用荧光显微镜检查Alexa488-CTb的注射部位。
    3. 将单个部分浸没在含有PB的10ml管中。
    4. 用0.25%无水乙酸盐在0.1M三乙醇胺-HCl(pH 8.0)中乙酰化切片10分钟 注意:在使用时准备解决方案。
    5. 在杂交缓冲液中预杂交1小时
    6. 在稀释度为1:1000的杂交缓冲液中补充地高辛(DIG)标记的cRNA探针,在63.5℃下进行12小时。
      注意:DIG标记的cRNA探针的制备在之前的我们的报告中描述(Kudo等人,2012; Yamasaki等人,2010)。杂交在杂交炉中进行。用石蜡膜覆盖管尖,以避免干燥。
    7. 在61℃下,用5×SSC洗涤30分钟,用含有50%甲酰胺(甲酰胺1)的4×SSC洗涤40分钟,用含有50%甲酰胺(甲酰胺2)的2×SSC洗涤40分钟,并用0.1×SSC洗涤15分钟。 br /> 注意:溶液在61°C的热水浴中预热,洗涤步骤在热水浴中进行。
    8. 在室温(RT)下,在0.1x SSC中孵育15分钟,在NTE缓冲液中20分钟,20mM碘酰胺在NTE缓冲液中孵育20分钟,NTE缓冲液孵育10分钟,并且TNT缓冲液孵育10分钟。 注意:所有后续步骤均在RT执行。
    9. 用DIG封闭缓冲液封闭切片30分钟,用0.5%TSA封闭缓冲液封闭切片30分钟
    10. 在DIG封闭缓冲液(1:1000)中在过氧化物酶缀合的抗DIG抗体中孵育2小时。
    11. 用TNT缓冲液洗涤15分钟两次。
    12. 使用Cy3-TSA plus扩增试剂盒系统(参见配方20)在阴影中进行10分钟检测过氧化物酶的信号。
    13. 用TNT缓冲液洗涤5分钟三次。
    14. 对于残留的过氧化物酶活性的失活,在TNT缓冲液中的3%H 2 O 2中孵育30分钟。
    15. 用TNT缓冲液洗涤5分钟三次。
    16. 使用荧光显微镜检查目标mRNA的荧光信号 注意:因为Alexa488-CTb的荧光变弱并且在杂交和杂交后洗涤后容易灭活,所以应当使用抗Alexa Fluor 488抗体通过免疫荧光检测示踪剂,如过程III所示。
    17. 对Alexa488-CTb进行免疫荧光后,用TOTO-3(PBS中1:50)复染20分钟。
    18. 在玻片上安装切片并使用荧光显微镜观察 注意:为了研究逆行追踪神经元的神经化学组成,收集平铺图像以覆盖整个示踪区域(x20放大倍数,x1.3缩放),并计数具有核的阳性细胞的数量(显示TOTO-3信号)(图2)。

      图2.组合的逆行示踪物标记和荧光原位杂交。将Alexa488-CTb注射到VTA中,并逆转运输到BST。在含有BST的切片中,进行Alexa Fluor 488(绿色)的免疫荧光,GAD mRNA(红色)的荧光原位杂交和TOTO-3(蓝色)的复染色。顶部图像是用于计数的平铺图像的一部分(x20放大率,x1.3缩放),底部图像是较高放大率(x60放大率,x1.0缩放)。共标记GAD mRNA和CTb(箭头)的神经元被鉴定为VTA-突出的GABA能神经元。比例尺:顶部,100μm;底部,30μm


  1. 盐水
    NaCl 9g/L dH 2 O 6/g
  2. 3.5%水合氯化
    水合氯醛1.75g/50ml dH 2 O
  3. 0.1 M PB
    制备1升0.1M PB
    将2.95g的NaH 2 PO 4·4·2H 2·2H 2 O和29g的Na 2 SO 4·2H 2 O混合, /sub>HPO4.12HO
    将dH 2 O加至1L并搅拌〜30分钟
  4. 5 N NaOH
    NaOH 40g/200ml dH 2 O
  5. 4%PFA
    加热500ml dH 2 O至80℃(不煮沸)
    加入40g PFA粉末,搅拌〜10分钟
    加入250μl5N NaOH,并保持搅拌直到溶液变得清澈
    接下来,在另一个烧杯中制备500ml的0.2M PB(溶液(b)) 将2.95g的NaH 2 PO 4·4·2H 2·2H 2 O和29g的Na 2 SO 4·2H 2 O混合, /sub>HPO4.12HO
    将dH 2 O加入到500ml中并搅拌〜30分钟
  6. PBS
    制备1升10x PBS储备液
    将87g的NaCl,3.1g的NaH 2 PO 4·4·2H 2·2H 2 O和28.7g的Na 2 HPO 4 12H 2 O
    将dH 2 O加入到1L中并搅拌〜2h
    使用dH 2 O稀释该10倍储备溶液1/10
  7. 20%Tween-20
    通过搅拌,混合20ml吐温-20和80ml ddH 2 O 存储在4°C
  8. 含有0.1%Tween-20(PBS-T)的PBS 加入2.5ml的20%吐温-20与500ml的PBS
  9. 0.25%无水乙酸在0.1M三乙醇胺-HCl(pH8.0)中的溶液 制备100ml乙酰化溶液
    将三乙醇胺-HCl与100ml dH 2 O混合 加入950微升5 N NaOH,确保pH为8.0
  10. 1 M Tris-HCl(pH 7.4或8.0)
    制备1 L Tris-HCl缓冲液
    将121.1g的Tris碱(三(羟甲基)氨基甲烷)与800ml的dH 2 O混合。 用盐酸将pH调至7.4或8.0 将dH <2> O添加到1 L
  11. 5 M NaCl
    制备1L的5M NaCl
    通过搅拌混合292.2g NaCl和800ml dH 2 O 将dH 2 O加至1L并搅拌直至它们完全溶解
  12. 0.5M乙二胺四乙酸(EDTA) 制备500ml 0.5M EDTA
    将93.1g EDTA与400ml dH 2 O混合 用5N NaOH将pH调至8.0 将dH <2> O添加到500ml
  13. 杂交缓冲区
    高压灭菌两个500毫升烧杯; 一个用250ml的dH 2 O 2填充,另一个是空的
    在烧杯冷却后,将80ml高压灭菌的dH 2 O和250ml甲酰胺倒入空烧杯中
    1 M Tris-HCl(pH 8.0)
    100 mg
    100x Denhardt *
    5 ml
    5M NaCl
    60 ml
    0.5 M EDTA
    1 ml
    * 100x Denhardt's
    数量(50 ml)
    dH 2 2 O 到50 ml
    将高压灭菌的dH 2 O加至500ml,然后振荡溶解〜24小时。
  14. 标准盐水柠檬酸盐(SSC)
    制备1 L 20x SSC储备液
    将175.3g NaCl和88.2g柠檬酸钠混合 加入dH2O至1L并搅拌〜2小时

    5x SSC
    0.1x SSC
    20x SSC
    250 ml
    5 ml
    30 ml
    15 ml
    75 ml
    75 ml
    dH 2 2 O  到1 L
    到1 L
  15. NaCl-Tris-EDTA(NTE)缓冲液 要生成1升NTE缓冲区
    混合100ml的5M NaCl,10ml的1M Tris-HCl(pH8.0),10ml的0.5M EDTA和25μl的20%Tween-20
    将dH <2> >添加到1 L并在RT保存
  16. Tris-NaCl-Tween(TNT)缓冲液
    将30ml 5M NaCl,100ml 1M Tris-HCl(pH7.4)和25μl20%Tween-20混合, 将dH <2> >添加到1 L并在RT保存
  17. 20mM碘酰胺的NTE缓冲液中 碘乙酰胺0.37g/100ml NTE缓冲液
  18. DIG封闭溶液
    正常绵羊血清:10%封闭试剂*:TNT缓冲液= 1:1:8
    * 10%封闭试剂(10ml)
    1. 制备马来酸缓冲液
      将58.1g马来酸和43.9g NaCl与400ml dH 2 O混合。 用5N NaOH将pH调节至7.5
    2. 用马来酸缓冲液溶解1g阻断试剂至10ml,振荡和加热
  19. 0.5%TSA封闭溶液
    向100毫升TNT缓冲液中加入0.5克封闭试剂 为了溶解封闭试剂,在搅拌下加热至60℃1小时
  20. Cy3-TSA扩增溶液
    将60μlDMSO加入Cy3扩增试剂(Cy3溶液;在4℃保存) 将此Cy3溶液稀释在1x Plus扩增稀释液(1:200)中。




  1. Kudo,T.,Uchigashima,M.,Miyazaki,T.,Konno,K.,Yamasaki,M.,Yanagawa,Y.,Minami,M.and Watanabe,M。(2012)。 三种类型的神经化学投影从成年小鼠的纹状终端的床核到腹侧被盖区 。 J Neurosci 32(50):18035-18046。
  2. Yamasaki,M.,Matsui,M.and Watanabe,M.(2010)。优先本地化 在树突状细胞轴上的毒蕈碱M1受体和皮质锥体细胞的脊柱及其传播的解剖学证据。 30(12):4408-4418。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Kudo, T. and Watanabe, M. (2013). Retrograde and Anterograde Tracing of Neural Projections. Bio-protocol 3(16): e870. DOI: 10.21769/BioProtoc.870.
  2. Kudo, T., Uchigashima, M., Miyazaki, T., Konno, K., Yamasaki, M., Yanagawa, Y., Minami, M. and Watanabe, M. (2012). Three types of neurochemical projection from the bed nucleus of the stria terminalis to the ventral tegmental area in adult mice. J Neurosci 32(50): 18035-18046.