Immunohistochemical Profiling of Neural Markers

Immunohistochemistry was performed as described (Rich et al., 2018 (link)). Primary antibodies were used against olfactory marker protein (Omp; goat, Wako 019-22291, Research Resource Identifier [RRID]: AB_664696; 1:500), Sox10 (rabbit, gift of Vivian Lee, Medical College of Wisconsin, WI, 1:3,000; Meng, Yuan, & Lee, 2011 ; Yardley & Garcia-Castro, 2012 (link)), Tbr1 (rabbit, Abcam ab31940, RRID: AB_2200219; 1:1,000; expression data summarized in File S3) and Tubb3 (neuronal αIII tubulin; mouse IgG2a, clone TUJ1, Covance MMS-435P, RRID:AB_2313773; 1:250). Matched AlexaFluor-conjugated secondary antibodies (Molecular Probes, Thermo Fisher Scientific) were used at 1:1,000. For triple immunostaining, anti-Tubb3 was detected by a biotinylated secondary antibody (goat antimouse IgG2a, Invitrogen, 1:100), followed by Alexa350-conjugated NeutrAvidin (Molecular Probes, 1:100). Slides were mounted with Fluoromount G (Southern Biotech, Birmingham, AL) or Vectashield with 4’,6-diamidino-2’-phenylindole dihydrochloride (DAPI; Vector Labs, Burlingame, CA).

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Perera S.N., Williams R.M., Lyne R., Stubbs O., Buehler D.P., Sauka-Spengler T., Noda M., Micklem G., Southard-Smith E.M, & Baker C.V. (2020). Insights into olfactory ensheathing cell development from a laser-microdissection and transcriptome-profiling approach. Glia, 68(12), 2550-2584.

Publication 2020

ab31940 MMS-435 AlexaFluor-conjugated secondary antibodies goat antimouse IgG2a Alexa350-conjugated NeutrAvidin Fluoromount G Vectashield with 4’,6-diamidino-2’-phenylindole dihydrochloride (DAPI;

Antibodies Antibody Clone Dapi Goat Igg2a Immunohistochemistry Molecular probes Mouse Neuronal Neutravidin Olfactory marker protein Rabbit Sox10 Tubb3 Tubulin Vector

Corresponding Organization : University of Cambridge

Other organizations : MRC Weatherall Institute of Molecular Medicine, University of Oxford, Vanderbilt University, National Institute for Basic Biology

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Variable analysis

independent variables

Primary antibodies used against olfactory marker protein (Omp), Sox10, Tbr1, and Tubb3 (neuronal αIII tubulin)

dependent variables

Immunohistochemistry staining patterns

control variables

Immunohistochemistry protocol as described in Rich et al., 2018
Matched AlexaFluor-conjugated secondary antibodies used at 1:1,000 dilution
Anti-Tubb3 detected by a biotinylated secondary antibody (goat anti-mouse IgG2a) followed by Alexa350-conjugated NeutrAvidin
Slides mounted with Fluoromount G or Vectashield with 4',6-diamidino-2'-phenylindole dihydrochloride (DAPI)

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