Journal: Genesis (New York, N.Y. : 2000)

Article Title: Quantitative assessment of the knockdown efficiency of morpholino antisense oligonucleotides in zebrafish embryos using a luciferase assay.

doi: 10.1002/dvg.20361

Figure Lengend Snippet: FIG. 1. Quantitative evaluation of the knockdown efficiency of sox2-MOs in zebrafish embryos using a MO-target luciferase assay. (Aa) Schematic representation of the sox2-luc fusion RNA construct used in the assay. The position of the zebrafish sox2 sequence is indicated by the dotted lines and was placed upstream of the lucif- erase gene. (Ab) The sequences of sox2-MO1, MO2, and MO3. (Ac) The nucleotide sequence of the zebrafish sox2 mRNA used in the fusion construct (2152 to 132). The MO target sequences are underlined and the start codon is also indicated by the boxed area. (B) Quantitative measurement of the inhibition levels caused by the injection of sox2-MOs using the luciferase assay. sox2-MO2 1 3 was a 1:1 mixture of sox2-MO2 and MO3. The luciferase activity generated by the sox2-luc fusion in the absence of MOs was arbi- trarily assigned a value of 100. Data are shown as the average val- ues of two independent injection experiments with standard errors. For one injection experiment, luciferase activity was derived from at least 21 injected embryos of the tail bud to early somite stages (see materials and methods). (C) Inhibition levels of endogenous SOX2 expression analyzed by western blotting. Lysates for SDS-PAGE were prepared from 40 embryos of the tail bud to early somite stages and a seven-embryo equivalent amount of proteins was used per lane. In the rightmost five lanes, twofold serial dilutions of the lysate from embryos without MO injection were loaded for com- parison purposes. Note that the anti-SOX2 antibody weakly cross- reacted with the other group B1 SOX proteins, SOX3, SOX19A, and SOX19B, which together comprise a faster migrating band below the SOX2 band. The identity of an upper band indicated by asterisk is unknown, but is possibly the result of a modification to SOX2 such as sumoylation (Tsuruzoe et al., 2006). As a loading control, the same blot was reprobed with anti-a-tubulin antibody (lower panel).

Article Snippet: To detect the SOX2 protein, anti-SOX2 antibody (AF2018, R&D systems, Minneapolis, MN) at 0.16 lg/ml in Can-Get-Signal immunoreaction enhancer solution (Toyobo, Osaka, Japan) was used.

Techniques: Knockdown, Luciferase, Construct, Sequencing, Inhibition, Injection, Activity Assay, Generated, Derivative Assay, Expressing, Western Blot, SDS Page, Control

Journal: Otology & Neurotology

Article Title: Selection Criteria Optimal for Recovery of Inner Ear Tissues From Deceased Organ Donors

doi: 10.1097/mao.0000000000003496

Figure Lengend Snippet: FIG. 1. Immunohistochemistry of utricles from human organ donors. A–D, Whole mount preparation of utricles were immunolabeled for the hair cell marker Myosin7a and the supporting cell markers Sox2. In utricles from organ donors #13 and #9, many hair cells and supporting cells were observed in (A and B). A’ and B’’’, Representative confocal images of the sensory epithelium at high magnification. A’’’’ and B’’’’) orthogonal views of A’ and B’. C and D, High and low magnification images showing no or few hair cells and a variable number of supporting cells in utricles from organ donors #4 and #7. E and F, In seven utricles from five organ donors, hair cell density was found to be 79.8 13.4 per 10,000 mm2 and supporting cell density 165.1 51.8 per 10,000mm2. Donor #9 is a cardiac death, from whom bilateral utricles showed the highest hair cell and supporting cell density (98.6 and 215.8 per 10,000 mm2, respectively). Donor #5 is a brain death donor, from whom the utricle from the right ear had the lowest hair cell and supporting cell densities (64.8 and 66.7 per 10,000 mm2, respectively). Each data point represents cell density from high magnification images (1–5 per utricle). Data shown as mean S.D., scale bars: A–D) 200 mm, A’ – D’) 20 mm.

Article Snippet: Antibodies against the following proteins were used: Myosin7a (1:1000, rabbit, #25–6790, Proteus Biosciences) and Sox2 (1:400, goat, #sc-17320, Santa Cruz Biotechnology or 1:200, goat, #AF2018, R&D).

Techniques: Immunohistochemistry, Immunolabeling, Marker