Expression of CTCFL and CTCF in the testis. A-C Immunohistochemical staining of testis sections. Paraffin-embedded sections from day 90 testes from heterozygous (del/+) and homozygous (del/del) Ctcfl mutant mice were stained with anti-CTCFL, followed by diaminobenzidine (DAB) coloring. Some of the CTCFL-positive cells are indicated with black arrowheads. Scale bars A, C: 100 μm, B: 50 μm. D-G Immunofluorescence staining of testis sections. Sections as described in A-C were stained with CTCFL (D and F) or STRA8 (E and G) antibodies. STRA8-positive cells in panels E and G are indicated with green arrowheads; the same cells are indicated with red arrowheads in the sections stained with anti-CTCFL antibodies (panels D and F). In Ctcfl mutant mice, STRA8 distribution is not changed. Scale bar is 50 μm. H-P Ex vivo confocal and multiphoton imaging of intact seminiferous tubules. Testis tubules were dissected from GFP-CTCFL- (H-M) or GFP-CTCF- (N-P) expressing mice, exposed to Hoechst at the adluminal side of the seminiferous tubule, and analyzed with a confocal/multiphoton microscope (GFP-CTCFL and GFP-CTCF, green; Hoechst, red). Panel H-J (see also Movie S1) shows a low magnification view of GFP-CTCFL distribution. Notice the presence of GFP-CTCFL-positive cells in the upper half of the tubule and their absence in the bottom half, indicating a transient population of cells. In (K-M) a high-magnification view of the same GFP-CTCFL-positive cells is shown. Notice the non-homogenous distribution of GFP-CTCFL in the nucleus. In (N-P) GFP-CTCF staining is shown. For clarity, some of the cell types are encircled, and their position is indicated in the other panels using white arrowheads. Pl = preleptone spermatocyte; rs = round spermatid; pa = pachytene spermatocyte; se = Sertoli cell. Bars, H-J: 70 μm, K-M: 10 μm, N-P: 25 μm.