Using in ovo electroporation, we devised a method to selectively transfect the auditory inner ear and cochlear nucleus in chicken embryos to achieve a cell-group-specific knockdown of fragile X mental retardation protein during discrete periods of circuit assembly.
Fragile X mental retardation protein (FMRP) is an mRNA-binding protein that regulates local protein translation. FMRP loss or dysfunction leads to aberrant neuronal and synaptic activities in fragile X syndrome (FXS), which is characterized by intellectual disability, sensory abnormalities, and social communication problems. Studies of FMRP function and FXS pathogenesis have primarily been conducted with Fmr1 (the gene encoding FMRP) knockout in transgenic animals. Here we report an in vivo method for determining the cell-autonomous function of FMRP during the period of circuit assembly and synaptic formation using chicken embryos. This method employs stage-, site-, and direction-specific electroporation of a drug-inducible vector system containing Fmr1 small hairpin RNA (shRNA) and an EGFP reporter. With this method, we achieved selective FMRP knockdown in the auditory ganglion (AG) and in one of its brainstem targets, the nucleus magnocellularis (NM), thus providing a component-specific manipulation within the AG-NM circuit. Additionally, the mosaic pattern of the transfection allows within-animal controls and neighboring neuron/fiber comparisons for enhanced reliability and sensitivity in data analyzing. The inducible vector system provides temporal control of gene editing onset to minimize accumulating developmental effects. The combination of these strategies provides an innovative tool to dissect the cell-autonomous function of FMRP in synaptic and circuit development.
Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by intellectual disability, sensory abnormalities, and autistic behaviors. In most cases, FXS is caused by a global loss of fragile X mental retardation protein (FMRP; encoded by Fmr1 gene) starting at early embryonic stages1. FMRP is an RNA-binding protein that is normally expressed in most neurons and glial cells in the brain, as well as in sensory organs2,3,4. In mammalian brains, FMRP is likely associated with hundreds of mRNAs that encode proteins that are important for various neural activities5. Studies of conventional Fmr1 knockout animals demonstrated that FMRP expression is particularly important to the assembly and plasticity of synaptic neurotransmission6. Several conditional and mosaic knockout models have further demonstrated that FMRP actions and signals vary across brain regions, cell types, and synaptic sites during several developmental events including axonal projection, dendritic patterning, and synaptic plasticity7,8,9,10,11,12,13,14. Acute function of FMRP in regulating synaptic transmission was studied by intracellular delivery of inhibitory FMRP antibodies or FMRP itself in brain slices or cultured neurons15,16,17,18. These methods, however, do not offer the ability to track FMRP misexpression-induced consequences during development. Thus, developing in vivo methods to investigate the cell-autonomous functions of FMRP is in great need, and expected to help determine whether the reported anomalies in FXS patients are direct consequences of FMRP loss in the associated neurons and circuits, or secondary consequences derived from network-wide changes during development19.
The auditory brainstem of chicken embryos offers a uniquely advantageous model for in-depth functional analyses of FMRP regulation in circuit and synapse development. The easy access to embryonic chicken brains and the well-established in ovo electroporation technique for genetic manipulation have contributed greatly to our understanding of brain development at early embryonic stages. In a recently published study, this technique was combined with advanced molecular tools that allow temporal control of FMRP misexpression20,21. Here, the methodology is advanced to induce selective manipulations of presynaptic and postsynaptic neurons separately. This method was developed in the auditory brainstem circuit. Acoustic signal is detected by hair cells in the auditory inner ear and then conveyed to the auditory ganglion (AG; also called the spiral ganglion in mammals). Bipolar neurons in the AG innervate hair cells with their peripheral processes and in turn send a central projection (the auditory nerve) to the brainstem where they terminate in two primary cochlear nuclei, the nucleus magnocellularis (NM) and the nucleus angularis (NA). Neurons in the NM are structurally and functionally comparable to the spherical bushy cells of the mammalian anteroventral cochlear nucleus. Within the NM, auditory nerve fibers (ANFs) synapse on the somata of NM neurons via the large endbulb of Held terminals22. During development, NM neurons arise from rhombomeres 5 and 6 (r5/6) in the hindbrain23, while AG neurons are derived from neuroblasts residing in the otocyst24. Here, we describe the procedure to selectively knockdown FMRP expression in the presynaptic AG neurons and in the postsynaptic NM neurons separately.
Eggs and chicken embryos were handled with care and respect in accordance with the animal protocols approved by the Jinan University Animal Care and Use Committee.
1. Egg and plasmid preparation
2. In ovo electroporation
3. Administration of Dox to initiate and maintain plasmid transcription
4. Tissue dissection and sectioning
5. Immunostaining and microscope imaging
NOTE: Two types of immunostaining are performed depending on whether sections are mounted on slides or free-floating in PBS.
By performing in ovo electroporation at different sites and at different developmental stages, we achieved selective FMRP knockdown either in the auditory periphery or in the auditory brainstem.
FMRP knockdown in NM
Small hairpin RNA (shRNA) against the chicken Fmr1 was designed and cloned into the Tet-On vector system as described previously20. The setup for in ovo electroporation is shown in Figure 1A. The plasmid DNA tinted with fast green was injected into the neural tube at the r5/r6 level at HH12 (Figure 1B–C). A platinum bipolar electrode was placed on each side of the neural tube at the r5/6 level (Figure 1D). Two pulses at 12 V were delivered to drive the plasmid into the tissue near the positive side of the electrode. Dox was applied on the chorioallantoic membrane to trigger the expression of Fmr1 shRNA and EGFP. After 24 h of Dox treatment, EGFP was evident under a fluorescent stereomicroscope. Figure 1E–F shows an example at E3 when Dox was applied at E2. Transverse sections confirmed the location of EGFP expressing (EGFP+) cells on one side of the hindbrain (Figure 1G–H). Additionally, a group of transfected cells was found anterior to the otocyst; these were cranial neural crest cells migrating ventrolaterally from the neural tube (Figure 1F). Electroporated embryos can also be incubated longer to study circuit formation at later stages. At E15, EGFP was observed in the brainstem, but only on the transfected side (Figure 1I–J). Cross sections at the level of the dorsal brainstem demonstrated a handful of EGFP+ neurons that were scattered in the NM on the side of electroporation (Figure 1L). EGFP+ fibers were seen projecting to the target of NM neurons: the dorsal nucleus laminaris (NL) on the ipsilateral side (Figure 1L) and the ventral NL on the contralateral side (Figure 1K). The knockdown effect of Fmr1 shRNA was validated by marked reductions in FMRP immunoreactivity in transfected NM neurons as compared to neighboring non-transfected neurons (Figure 1M–N). In the auditory ganglion (AG), neurons were non-transfected (EGFP-), although some glial cells surrounding AG neurons were EGFP+ (Figure 1O–P), presumably derived from EGFP+ cranial neural crest cells (see Figure 1F). Thus, this electroporation strategy provides a selective transfection of the postsynaptic neurons in the AG-NM circuit.
FMRP knockdown in the auditory duct
The plasmid mixture was injected into the otocyst at HH13 (Figure 2A), followed by electroporation by placing the positive side of the electrode anterior to the otocyst and the negative side posterior to the otocyst (Figure 2B). Whole-head sections at E6 demonstrated strong EGFP fluorescence in the right otocyst, but not in the brainstem (Figure 2C). Isolated auditory ducts at E9 exhibited extensive EGFP fluorescence throughout the length of the duct (Figure 2D–E). On transverse sections, EGFP+ cells were confirmed in the basilar papilla (BP) along the wall of the cochlear duct and in the AG (Figure 2F–G). The knockdown effect of Fmr1 shRNA was validated by marked reductions in FMRP immunoreactivity in transfected hair cells (*, Figure 2H–J) as compared to neighboring non-transfected hair cells (•, Figure 2H–J). For supporting cells, FMRP immunoreactivity was weak in both transfected and non-transfected cells (Figure 2H–J). Similar to hair cells, FMRP immunoreactivity was largely diminished in transfected AG neurons as compared to neighboring non-transfected neurons (Figure 2K–M).
We next tracked the central projection of transfected AG neurons to the brainstem via the auditory nerve. The mosaic pattern of Fmr1 shRNA transfection in the AG was further confirmed at E6 using Islet-1 immunoreactivity (Figure 3A), a marker for the developing chicken inner ear31. When a part of the AG was extracted with the brainstem during dissection, isolated EGFP+ AG neurons and their fibers within the brainstem were visualized in situ in the same preparations (Figure 3B–C). On transverse sections at the level of the NM, EGFP+ ANFs had reached the NM at E9 (Figure 3D) and were continuously observed at E15 and E19 (Figure 3E–F). High-magnification images revealed growth cone-like endings of ANFs at E9 (Figure 3H), which formed the large palm-like endbulb at E15 before they grew into the characterized endbulb of Held morphology with complex branches at E19 (Figure 3I–J). Beyond the NM, extensive branches of ANFs were also seen in the NA, another primary cochlear nucleus (Figure 3E). This was expected, as the same ANFs bifurcate to innervate both the NM and the NA in chickens32. We also saw EGFP+ fibers entering the adjacent vestibular cell groups, including the descending vestibular nucleus (VeD) as shown in Figure 3E. Although we optimized the location of the electroporating electrode to preferentially target the anterior portion of the otocyst where AG is formed, some vestibular ganglion neurons were also transfected. Immunostaining for parvalbumin, a marker for ANFs in chickens, can be used to differentiate auditory vs. vestibular fibers in the brainstem (Figure 3F–G).
In summary, this electroporation strategy provides a selective transfection of the presynaptic neurons in the AG-NM circuit and can be used to track the development of endbulb of Held at individual terminal levels following genetic manipulations.
Figure 1. FMRP knockdown in the chicken NM. (A) Image of electroporation setup. (B) Schematic drawing showing the microinjection site at the rhombomere r5/6 level of an HH12 chicken embryo. Abbreviations: NT = neural tube; SO = somite. (C) For enhanced visibility, Indian ink (black) was injected beneath the embryo with a syringe. Plasmid mixture tinted with fast green was then injected into the lumen of the neural tube. (D) Positioning of the bipolar electrode for electroporation. + and – indicate the positive and negative sides, respectively. Note, the blue plasmid mixture diffused into the neural tube on the positive side after electroporation. (E–F) Bright field (BF) image (E) merged with EGFP channel (F) of an electroporated chicken embryo at embryonic day 3 (E3), showing transfection in the neural tube at the same anteroposterior level of the otocyst. Dox was applied at E2, 6 h following the electroporation. Migrating cranial neural crest (CNC) cells are indicated by the yellow arrow and magnified in the inset in F. Scale bar = 500 µm in E, applies to E and F; 100 µm in the inset. (G–H) Transfected cells with Fmr1 shRNA and EGFP on the cross section of a transfected E3 embryo. EGFP+ cells were confined on one side of the neural tube. The section was double stained with FMRP immunoreactivity (gray). Scale bar = 40 µm in G, applies to G and H. (I–J) BF image (I) merged with EGFP channel (J) of an E15 brainstem following Dox treatment at E8. Scale bar = 1 mm in I, applies to I and J. (K–L) Cross section of an E15 brainstem with EGFP+ NM neurons on the transfected (trans) side only. On the contralateral (contra) side (K), EGFP+ axons were seen in the ventral portion of the nucleus laminaris (NL). (M–N) High magnification images of the NM at E19 with double labeling of FMRP (red) and SNAP25 (blue) immunostaining. SNAP25 is a presynaptic marker that labels the endbulb of Held. Note the marked reduction of FMRP immunoreactivity in the transfected (green; *) neurons as compared to neighboring non-transfected neurons (•). (O–P) High magnification images of the auditory ganglion (AG) at E19 with FMRP immunoreactivity. AG neurons (AGN) were not transfected (EGFP-). Some glial cells derived from CNC cells indicated in F were transfected. Scale bar = 10 µm in M, applies to M-P. Please click here to view a larger version of this figure.
Figure 2. FMRP knockdown in the chicken auditory duct. (A) Bright field (BF) image showing the microinjection of plasmids into the otocyst at HH13. Indian ink (black) was injected below the embryo for enhanced visibility. (B) Positioning of the bipolar electrode for electroporation. The positive side (labeled with a plus symbol, +) was positioned anterior to the otocyst, and the negative side (labeled with a minus symbol, -) was placed posterior to the otocyst. (C) Transverse section of a chicken head at E6 was immunostained for FMRP (red) and neurofilament (NF; blue). Dox was applied at E3 following the electroporation. EGFP fluorescence was detected in the otocyst but not in the brainstem. Scale bar = 100 µm. (D–E) BF image (D) merged with EGFP channel (E) of an auditory duct at E9. Scale bar = 500 µm. (F–G) Transverse section of an E9 auditory duct with FMRP (red) and NF (blue) immunostaining. EGFP+ cells were seen in the cochlear duct wall, basilar papilla (BP), and auditory ganglion (AG). Scale bar = 50 µm in F, applies to F and G. (H–J) High magnification images of an E9 BP showing largely diminished FMRP immunoreactivity in transfected hair cells (HCs; *) compared to non-transfected HCs (•). NF-positive fibers (blue) were the periphery innervation from AG neurons. (K–M) High magnification images of AG neurons at E9 with FMRP immunostaining (red). FMRP immunoreactivity was strong in non-transfected neurons (•) and not detectable in transfected neurons (EGFP+; *). Scale bar = 50 µm in H, and applies to H-M. Please click here to view a larger version of this figure.
Figure 3. Axonal tracking of transfected AG neurons in the brainstem. Embryos were electroporated at HH13 for otocyst transfection of Fmr1 shRNA. Dox application started at E2. (A) Transverse sections of an E6 chicken head with Islet-1 immunostaining and DAPI counterstain. EGFP+ cells were evident in the auditory ganglion (AG). Stars* indicate several transfected AG neurons in the inset. Scale bar = 100 µm in A and 10 µm in inset. (B–C) Bright field (BF) image (B) merged with EGFP channel (C) of an E9 brainstem. In this case, a portion of the AG (white arrows) was left connected to the auditory brainstem. Yellow arrow indicates transfected AG neurons (AGN) in the inset. EGFP+ fibers were evident on the transfected side of the brainstem. Scale bar = 1 mm in B, applies to B and C; 100 µm in the inset. (D) Cross section of the E9 brainstem at the level indicated in C by the dashed line. EGFP+ auditory nerve fibers (ANF; green) extended along the dorsal margin of the brainstem and approached the nucleus magnocellularis (NM). Scale bar = 100 µm. (E–G) EGFP+ fibers at E15 (E) and E19 (F–G). Some fibers were also seen in the nucleus angularis (NA) and the descending vestibular nucleus (VeD). E19 sections were stained for parvalbumin (PV) immunoreactivity as a marker for ANFs. The dashed box in F is magnified in the insets, showing an EGFP+ ANF, which was PV immunoreactive. Scale bar = 100 µm in E; 50 µm in F, applies to F and G; 2 µm in the insets. (H–J) High magnification images of ANF terminals in NM at E9, E15, and E19, showing the formation and progressive maturation of the endbulb of Held. Scale bar = 5 µm in H, applies to H-J. Please click here to view a larger version of this figure.
To determine the cell-autonomous function of FMRP, manipulating its expression in individual cell groups or cell types is necessary. Since one of the major functions of FMRP is to regulate synaptic formation and plasticity, selectively manipulating each synaptic component of a certain circuit is prerequisite for a full understanding of FMRP mechanism in synaptic communication. Using in ovo electroporation of chicken embryos, we demonstrated a method to target FMRP expression in the AG-NM circuit, either in presynaptic AG neurons or postsynaptic NM neurons. To achieve this, choosing appropriate developmental stages for electroporation and accurately positioning the electroporation electrode on the embryo are critical steps. NM neurons arise from neural progenitors in the neural tube at the r5/6 level at HH1223. At the same stage, cranial neural crest cells migrate from the dorsal tip of the neural tube to the cochleovestibular ganglion to envelop future AG neurons33. Thus, both the NM neurons and the glia cells in the AG are neural tube derived. In contrast, hair cells and AG neurons originate from the ectoderm24. The ectoderm adjacent to the r5/6 level thickens at HH10 and then invaginates to form the otic cup at HH13. The otic cup then closes and separates from the ectoderm to form the otocyst, with the anterior portion becoming the auditory sensory organ and the posterior portion contributing to the vestibular system. In both the auditory and vestibular portions, the neuroblast cells residing in the ventral part of the otic cup delaminate and migrate to form the cochleovestibular ganglion. Building upon these fate mapping studies, we transfected the progenitors of NM neurons by electroporating the neural tube at HH12, a strategy that has been established previously26,28,34. Although this strategy leads to additional transfection of glial cells in the brainstem and the AG, none of the AG neurons were transfected. Neuronal-specific transfection can be achieved by employing an Atoh1 promotor-driven strategy, as what we did previously21. For selective transfection of AG neurons, we electroporated the otocyst at HH13. Plasmids injected into the otic cup predominantly entered the anterior portion of the otocyst for hair cell and AG neuron transfection simultaneously when positioning electrodes as shown in Figure 2B. To preferentially target AG neurons over hair cells, it is feasible to inject the plasmid solution straight into the region adjacent to the anterior otocyst, where the delaminated neuroblasts locate35. However, this method has a lower efficiency of AG neuron transfection as compared to otocyst injection, as the plasmids diffuse quickly after injection. This method may also lead to additional transfection of the surrounding head mesenchymal cells. One method to preferentially target hair cells over AG neurons is to delay the otocyst injection and electroporation to HH18 (which is ~embryonic day 3), as the majority of neuroblasts (AG neuron precursors) have migrated out from the otocyst at this stage24.
With modifications, this method can be extended to study the vestibular system. For vestibular ganglion transfection, the direction of the electrodes shown in Figure 2B must be reversed to transfect the posterior portion of the otocyst due to the distinct location of auditory and vestibular ganglion precursors.
In ovo electroporation of chicken embryos is a well-established technique for investigating early stages of development (within the first week of incubation). For long-term studies, survival rate is a major concern. When starting Dox treatment at E8, the survival rate found here is ~60% at E9, but drops to 30% at E15, and even lower at E19. Hatchlings from electroporated eggs are possible but rare and can only survive for several days in most cases. To increase the survival rate, the following should be considered: 1) use an electroporator that provides square waves instead of sharp waves; 2) apply one or two drops of sterile PBS to the top of the embryo if the albumin layer is not thick enough to promote electric conductivity (this will help reduce electrical trauma); 3) opening of the vitelline membrane to expose the embryo at the injection site is not required for a successful transfection and may harm the embryo; 4) to reduce the risk of contamination, use a syringe to poke the parafilm and administer Dox instead of reopening the window. After injection, wipe the transparent film with 75% ethanol and cover the needle opening with tape; 5) for otocyst transfection, inject plasmids into the otocyst dorsolaterally with a 45° angled pipette to avoid possible puncture of the aorta dorsalis underneath. If the aorta dorsalis is damaged, bleeding will flush out the plasmids and even cause death; 6) if possible, avoid using Indian ink to visualize the embryo. Less handling and fewer procedures will result in a higher survival rate.
In addition to the ability to selectively transfect AG or NM neurons, in ovo electroporation provides a unique system that allows neighboring comparisons for data analyses. After electroporation, only a subset of cells in either the NM or the AG are transfected, allowing the surrounding non-transfected cells in the same cell group to serve as an ideal control20. If potential interaction among neighboring neurons is a concern, neuron counterparts from the non-transfected side of the ear and brain can serve as an additional control. For histological and imaging studies, this outcome enables data analyses with high efficiency at the single cell or single fiber level. Molecular analyses are also feasible if combined with fluorescent-based cell sorting and large-scale protein and RNA analyses such as mass spectrometry and next-generation sequencing. However, using in ovo electroporation as a means of gene editing may be challenging for functional and behavior studies because the percentage of transfected cells within a cell group may not be large enough to result in functional consequences.
It is essential to perform two verification analyses when adopting the in ovo electroporation approach. First, the effect of gene editing on the protein of interest should be confirmed. In ovo electroporation transfects only a subset of neurons in the NM or AG (i.e., a mosaic pattern). Performing western blot on homogenized tissues that contain a mixture of transfected and non-transfected neurons is not sensitive enough to accurately reflect the knockdown effect. Thus, the validation must be conducted at the individual cell level using methods such as immunocytochemistry. For this purpose, we previously generated an antibody that recognizes chicken FMRP. The specificity of this antibody was verified by immunocytochemistry and western blot in a previous study30. The knockdown effect of Fmr1 shRNA was validated quantitatively by observing significant reductions in the intensity of FMRP immunoreactivity in transfected NM neurons as compared to neighboring non-transfected neurons. Second, control groups using scrambled RNAs, or other control constructs, should be employed to confirm the specificity of observed cellular effects of FMRP misexpression20,21. To achieve this goal, we designed a scrambled shRNA, cloned it into the same Tet-On vector system, and electroporated it into NM precursors, as described previously20. FMRP expression in the NM neurons transfected with the scrambled shRNA was not affected, as evidenced by the unchanged intensity of FMRP immunoreactivity as compared to neighboring non-transfected neurons. We have also identified a number of effects of Fmr1 shRNA transfection on axonal growth, dendritic pruning, terminal formation, and neurotransmission (reviewed in Curnow and Wang, 2022)36. We concluded that these effects were induced specifically by cell autonomous loss of FMRP because the scrambled shRNA failed to induce similar changes.
In conclusion, the in ovo electroporation technique enables selective Fmr1 gene editing with temporal control and component-specificity in the auditory ear-brainstem circuits and can be modified to manipulate the vestibular system. Development of this advanced technology in the chicken embryo, a well-established model in developmental biology, has and will continue to contribute to our understanding of brain development under normal and abnormal conditions such as FXS.
The authors have nothing to disclose.
This study was supported by: a National Natural Science Foundation of China grant (No. 32000697); the Science and Technology Program of Guangzhou (202102080139); the Guangdong Natural Science Foundation (2019A1515110625, 2021A1515010619); the Fundamental Research Funds for the Central Universities (11620324); a Research Grant of Key Laboratory of Regenerative Medicine, Ministry of Education, Jinan University (No. ZSYXM202107); the Fundamental Research Funds for the Central Universities of China (21621054); and the Medical Scientific Research Foundation of Guangdong Province of China (20191118142729581). We thank the medical experimental center of Jinan University. We thank Dr. Terra Bradley for careful editing of the manuscript.
Egg incubation | |||
16 °C refrigerator | MAGAT | Used for fertilized egg storage. | |
Egg incubator | SHANGHAI BOXUN | GZX-9240MBE | |
Fertilized eggs | Farm of South China Agricultural University | Eggs must be used in one week for optimal viability. | |
Plasmid preparation | |||
Centrifuge | Sigma | 10016 | |
Fast green | Solarbio | G1661 | Make 0.1% working solution in distilled water and autoclave. |
Plasmid Maxi-prep kit | QIAGEN | 12162 | Dissolve plasmid DNA in Tris-EDTA (TE) buffer; endotoxin-free preparation kit |
Sodium Acetate | Sigma-Aldrich | S2889 | Make 7.5M working solution in nuclase-free water. |
Electroporation and Doxycycline Administration | |||
Electroporator | BTX | ECM399 | |
1 mL / 5 mL Syringe | GUANGZHOU KANGFULAI | ||
Dissecting microscope | CNOPTEC | SZM-42 | |
Doxcycline | Sigma-Aldrich | D9891 | Use fresh aliquots for each dose and store at -20 °C. |
Glass capillary | BEIBOBOMEI | RD0910 | 0.9-1.1 mm*100 mm |
Laboratory parafilm | PARAFILM | PM996 | transparent film |
Pipette puller | CHENGDU INSTRUMENT FACTORY | WD-2 | Pulling condition: 500 °C for 15 s |
Platinum elctrodes | Home made | 0.5 mm diameter, 1.5 mm interval. | |
Platinum elctrodes | Home made | 0.5 mm diameter, 1.5 mm interval. | |
Rubber tube | Sigma-Aldrich | A5177 | |
Tissue Dissection and Fixation | |||
Forceps | RWD | F11020-11 | Tip size: 0.05*0.01 mm |
Other surgery tools | RWD | ||
Paraformaldehyde | Sigma-Aldrich | 158127 | Freshly made 4% PFA solution in phosphate-buffered saline can be stored in 4 °C for up to 1 week. |
SYLGARD 184 Silicone Elastomer Kit | DOW | 01673921 | For black background plates, food-grade carbon powder is applied. |
Sectioning | |||
Cryostat | LEICA | CM1850 | |
Gelatin | Sigma-Aldrich | G9391 | From bovine skin. |
Sliding microtome | LEICA | SM2010 | |
Immunostaining | |||
Alexa Fluor 488 goat anti-Mouse | Abcam | ab150113 | 1:500 dilution, RRID: AB_2576208 |
Alexa Fluor 555 goat anti-rabbit | Abcam | ab150078 | 1:500 dilution, RRID: AB_2722519 |
DAPI | Abcam | ab285390 | 1: 1000 dilution |
Fluoromount-G mounting medium | Southern Biotech | Sb-0100-01 | |
FMRP antibody | Y. Wang, Florida State University | #8263 | 1:1000 dilution, RRID: AB_2861242 |
Islet-1 antibody | DSHB | 39.3F7 | 1:100 dilution, RRID: AB_1157901 |
Netwell plate | Corning | 3478 | |
Neurofilament antibody | Sigma-Aldrich | N4142 | 1:1000 dilution, RRID: AB_477272 |
Parvalbumin antibody | Sigma-Aldrich | P3088 | 1:10000 dilution, RRID: AB_477329 |
SNAP25 antibody | Abcam | ab66066 | 1:1000 dilution, RRID: AB_2192052 |
Imaging | |||
Adobe photoshop | ADOBE | image editing software | |
Confocal microscope | LEICA | SP8 | |
Fluorescent stereomicroscope | OLYMPUS | MVX10 | |
Olympus Image-Pro Plus 7.0 | OlYMPUS | commercial image processing software package |