Multimodal profiling of neuronal cell types

Hybridization chain reaction (HCR) and Whole Mount Immunohistochemistry were done in accordance with previous described methods [42 (link), 61 (link)]. The HCR probes transcripts were synthesized by Molecular Instruments for ret, NM_181662.2; gfra1a, NM_131730.1; oprl1, NM_205589.2; oprd1b, NM_131258.4; etv1, XM_005157634.4; elavl3, NM_131449. The following primary antibodies were used: goat polyclonal IgG anti-Choline Acetyltransferase (ChaT, Millipore Sigma, AB144P, 1:500), rabbit polyclonal IgG anti-5-HT (serotonin, Immunostar, 20080, 1:250), mouse monoclonal IgG2b anti-HuC/D (Invitrogen Thermo Fisher, A-21271, 1:250), Mouse monoclonal IgG1 anti-Phox2b (B-11, Santa Cruz Biotechnology, SC-376997, 1:250). The following secondary antibodies were used from Invitrogen: Alexa Fluor 488 donkey anti-goat IgG (A-11055, 1:600), Alexa Fluor 405 goat anti-rabbit IgG (A-48254, 1:600), Alexa Fluor 647 goat anti-mouse IgG2b (A-21242, 1:600), Alexa Fluor 594 goat anti-mouse IgG1 (A-21125, 1:600). High content semi-automated confocal imaging and processing was done as described above.

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Moreno-Campos R., Singleton E.W, & Uribe R.A. (2024). A targeted CRISPR-Cas9 mediated F0 screen identifies genes involved in establishment of the enteric nervous system. PLOS ONE, 19(5), e0303914.

Publication 2024

gfra1a oprl1 oprd1b elavl3 mouse monoclonal IgG2b anti-HuC/D Mouse monoclonal IgG1 anti-Phox2b (B-11, Alexa Fluor 488 donkey anti-goat IgG Alexa Fluor 405 goat anti-rabbit IgG Alexa Fluor 647 goat anti-mouse IgG2b Alexa Fluor 594 goat anti-mouse IgG1

Corresponding Organization : Rice University

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Variable analysis

independent variables

HCR probes for ret, gfra1a, oprl1, oprd1b, etv1, elavl3

dependent variables

Expression of ret, gfra1a, oprl1, oprd1b, etv1, elavl3 transcripts
Immunoreactivity for ChAT, 5-HT, HuC/D, Phox2b

control variables

Adherence to previously described methods for Hybridization Chain Reaction (HCR) and Whole Mount Immunohistochemistry

controls

No positive or negative controls were explicitly mentioned in the provided information.

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