Journal: Biology of reproduction

Article Title: Effects of TGFbeta2 on wild-type and Tgfbr3 knockout mouse fetal testis.

doi: 10.1095/biolreprod.112.102194

Figure Lengend Snippet: FIG. 5. Assessment of mRNA expression levels of somatic cell genes in fetal testis explants after TGFb2 treatment. A–F) The mRNA expression levels of the genes encoding Nr5a1 (A), Star (B), Cyp11a1 (C), Hsd3b1 (D), Cyp17a1 (E), and Insl3 (F) in wild-type and Tgfbr3 knockout explants were analyzed by qRT-PCR and normalized to 18S rRNA levels. Means 6 SD of triplicate measurements of expression within single explants (n ¼ 5 explants/genotype/ treatment) are shown.*P , 0.05, **P , 0.01, ***P , 0.001. G–J) Images of sections through testis explants that have been immunostained for CYP11A1. Black arrowheads indicate Leydig cells. Int, interstitium. Dotted lines denote the boundary of representative cords (Co). Bar ¼ 50 lm.

Article Snippet: Antigen retrieval was performed in 50 mM glycine pH 3.5, maintained at 908C for 10 min. A monoclonal primary antibody against INHA (inhibin alpha, 1:800; gift of Professor David Robertson, Prince Henry’s Institute, Australia) or polyclonal primary antibodies against TGFb2 (sc-90, 1:50; Santa Cruz), CYP11A1 (also known as P450-cholesterol side chain cleavage) (ab1294, 1:800; Abcam), AMH (also known as MIS) (sc-6886, 1:400; Santa Cruz), SOX9 (sc-20095, 1:100; Santa Cruz), or DDX4 (also known as MVH) (ab13840, 1:800; Abcam), or laminin polyclonal antibody (L9393, 1:1000; Sigma) were applied for overnight incubation in 0.1% bovine serum albumin/phosphate buffered solution (PBS).

Techniques: Expressing, Knock-Out, Quantitative RT-PCR