After 10–12-day in vitro, neurons were fixed for 10 minutes in 4% paraformaldehyde in 0.12 M phosphate-buffered saline (PBS), washed 3 times in PBS, and blocked in 1% bovine serum albumin (Sigma), 0.1% Triton (Sigma) in PBS for 1 hour, then incubated with primary antibodies against either NRG4 (1:200, goat-polyclonal, Santa Cruz), ErbB4 (1:200, rabbit or mouse-monoclonal, Abcam, Cambridge, MA), DARPP-32 (1:400, rabbit-monoclonal, Cell Signaling Technology, Danvers, MA) or βIII-tubulin (1:500, mouse-monoclonal, R&D Systems) at 4°C overnight. After washing, the neurons were incubated with polyclonal Alexa-conjugated secondary antibodies (donkey anti-goat Alexa-546, donkey anti-rabbit Alexa-488, Invitrogen) 1:500 for 1 hour at room temperature. Cells were then washed, incubated with DAPI (1:8000) and visualized using a Zeiss LSM710 confocal microscope. For double immunolabeling, the cultures were incubated simultaneously with both primary and secondary antibodies.
Erbb4
Manufactured by Abcam
ErbB4 is a receptor tyrosine kinase protein. It is a member of the ErbB family of receptors.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (6 mentions)
Alexa conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
β 3 tubulin, by R&D Systems (1 mentions)
Darpp 32, by Cell Signaling Technology (1 mentions)
Pdgfra, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
Fluorescently conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti erbb2, by Abcam (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Fluoromount g, by Electron Microscopy Sciences (1 mentions)
Brachyury, by R&D Systems (1 mentions)
Iba 1, by Abcam (1 mentions)
Ang 2, by Abcam (1 mentions)
Cxcl10, by Thermo Fisher Scientific (1 mentions)
Brain derived neurotrophic factor (bdnf), by Thermo Fisher Scientific (1 mentions)
βiii tubulin tuj1, by BioLegend (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
6 protocols using erbb4
1
Immunolabeling of Neuronal Markers
2
Immunostaining of Neuronal Proteins
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Neurons were fixed for 10 mins in 4% paraformaldehyde in 0.12 M phosphate-buffered saline (PBS), washed 3 times in PBS and blocked in 1% BSA (Sigma), 0.1% Triton (Sigma) in PBS for 1 h, then incubated with primary antibodies (1:50) against NRG4 (Santa Cruz), ErbB4 (Abcam), Emx1 (Santa Cruz) overnight at 4 °C. After washing, the neurons were incubated with polyclonal Alexa-conjugated secondary antibodies (Invitrogen) 1:500 for 1 h at room temperature. Cells were then washed, incubated with DAPI (1:8000) and visualized using a Zeiss LSM710 confocal microscope.
3
Western Blot Analysis of ERBB4 and PDGFRA
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Cells were lysed in ice using RIPA buffer containing the protease inhibitor PMSF (Auragene, China). The protein concentration was then measured with BCA kit (Auragene, China). Primary antibodies against ERBB4 (1:1,500, Abcam, Cambridge), PDGFRA (1:1,000, Abcam, Cambridge), and β-actin (1:1,000, Abcam, Cambridge) were incubated with the film at 4 °C overnight. Next, the film was incubated in goat anti-mouse/rabbit IgG (H + L)-HRP polymer (1:15,000, Auragene, China) for 40 minutes, and then exposed to ECL chromogenic solution (Auragene, China). Finally, Image-Pro Plus 6.0 software was used to quantitatively analyze the intensity of the protein bands.
4
ErbB Receptor Visualization on Axons
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Example 10
Visualization of ErbB Receptor on Axons
To visualize ErbB receptor expression on axons, DRGs were cultured as described above for the screen. After 24 hours of growth, cells were fixed by adding 8% PFA/30% sucrose to culture medium at a 1:1 ratio for 30 minutes and washed 1× with PBS. Fixed cells were incubated in 5% BSA and 0.2% TritonX100 in PBS for 30 minutes, and then incubated overnight in 2% BSA in PBS at 4° C. with the following antibodies: (1) 50 μg/ml anti-EGFR (D1-5, Genentech), (2) 1:500 anti-ErbB2 (Abcam), (3) 24 μg/ml anti-ErbB3 (57.88, Genentech), and (4) 1:500 ErbB4 (Abcam). Cells were washed 1× with PBS, followed by incubation with a fluorescently conjugated secondary antibody (1:200, Invitrogen) at room temperature for 30 minutes, washed 1× with PBS containing Hoechst 33258 (1 μg/ml, Invitrogen), followed by a final PBS wash, and coverslipped with 250 μl of Fluoromount G (Electron Microscopy Sciences). As shown in
5
Histological Analysis of Cortical Organoids
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Brain cortical organoids at 20-100 days in culture were fixed with 10% formalin, dehydrated with ethanol, and embedded in paraffin blocks. 10 μm thick sections were deparaffinized with xylene, rehydrated in a graded series of ethanol and double distilled water in a standard manner, and then stained with H&E. To detect protein expression, the sections were heated to unmask the epitopes, blocked with 10% goat serum, then primary antibodies were added overnight [Sox2 from EMD Millipore, β III tubulin (TUJ1, BioLegend, San Diego, CA)]; IBA-1, NRG1, ErbB4, GFAP, CXCR3, Ang2 and Ang1 from Abcam; Brachyury from R&D Systems; Cleaved caspase 3 (CC3), NFL and NFM from Cell Signaling, Danvers, MA; BDNF from Thermo Fisher Scientific and CXCL-10 from PeproTech (Rocky Hill, NJ). Fluorescence staining was performed using fluorochrome-labelled secondary antibodies (Alexa Fluor 488, Alexa Fluor 647, Invitrogen). The sections were covered with Vectashield mounting medium containing DAPI (H-1200) obtained from Vector Laboratories Inc (Burlingame, CA).
6
Immunostaining of Brain Sections and Neurons
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Brain sections or glass coverslips were xed with 4% paraformaldehyde for 30 min, rupture of cell membrane was performed using 0.1% Triton X-100 for 10 min at room temperature, PBS containing 5% normal goat serum for 1 h at room temperature was used for blocking, and sections or coverslips were incubated overnight at 4 °C with the following primary antibodies: ErbB4, NRG, FGF2, Sirt1, syntaxin, Homer, and MAP2 (Abcam). Alexa Fluor 488 (green)/Alexa Fluor 594 (red) conjugated secondary antibody (Abcam, Cambridge, MA) were then used to detect primary antibodies for 1 h.
For dendritic spine analysis, the primary neurons coverslips were incubated with the primary antibodies: microtubule-associated protein 2B (MAP2B; 1:200; BD Transduction Laboratories, San Jose, CA, USA) and vesicular glutamate transporter 1 (vGlut1; 1:100; Neuromab, Davis, CA, USA) overnight at 4 °C. Alexa Fluor 488 (green)/Alexa Fluor 594 (red) conjugated secondary antibody (Abcam, Cambridge, MA) were then used to detect primary antibodies for 1 h. At least 10 cultured primary neurons per coverslip were used for quantitative analysis.
For dendritic spine analysis, the primary neurons coverslips were incubated with the primary antibodies: microtubule-associated protein 2B (MAP2B; 1:200; BD Transduction Laboratories, San Jose, CA, USA) and vesicular glutamate transporter 1 (vGlut1; 1:100; Neuromab, Davis, CA, USA) overnight at 4 °C. Alexa Fluor 488 (green)/Alexa Fluor 594 (red) conjugated secondary antibody (Abcam, Cambridge, MA) were then used to detect primary antibodies for 1 h. At least 10 cultured primary neurons per coverslip were used for quantitative analysis.
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