Amersham cell proliferation labeling reagent

BrdU (5-Bromo-2-deoxyridine) incorporation assay was used to quantitate cell cycle entry and proliferation. 10 μl of BrdU (Amersham Cell Proliferation Labeling Reagent, GE Healthcare Life Sciences, Little Chalfont, UK) per gram body weight was administered via IP on alternate days following UUO. Double immunostaining was performed for BrdU and tdTomato. Antibody paraffin embedded tissue was prepared and blocked as described above. Avidin/biotin blocking (Vector Laboratories, Burlingame, CA, USA) was performed to block endogenous biotin and prevent unspecific staining while using biotin-streptavidin labeling system. Tissue was incubated overnight at 4 °C with a primary mouse anti-BrdU (1:200, Amersham, GE Life Sciences, Buckinghamshire, UK). This was followed with a biotinylated goat anti-mouse antibody (1:500, Jackson Immunoresearch Laboratories, Inc, West Grove, PA, USA) incubated at room temperature for 1 h. The signal was amplified by incubation with streptavidin-conjugated with Alexa Fluor 488 (1:100; Invitrogen, Grand Island, NY, USA) for 45 min. Negative control staining was performed by omitting primary antibody staining.

P5 neonates were given 10 μL of Amersham Cell Proliferation Labeling Reagent (GE Healthcare) per gram of body weight via i.p. injection and testes were isolated 5 days later. The tunica albuginea of the testis was removed, and seminiferous tubules were mechanically separated and fixed in 4% paraformaldehyde (Sigma) for 5 h at 4°C. Fixed tubules were denatured with 3.5 M HCl for 2 min and blocked (2% BSA, 10% fetal bovine serum, 0.3% Triton X-100 in PBS) for 4 h at RT. Tubules were incubated with primary antibodies overnight at 4°C followed by secondary antibodies for 1.5 h at RT. Coverslip was mounted using DAPI-containing Hydromount. At least 1 cm of tubule was analyzed. A_single cells were cells that stood alone or if cytoplasmic connections were present, cells that were more than one nuclear length apart. An A_single event was counted when an A_single cell was spotted or when two A_single cells were found in close proximity (within the counting frame). A_paired cells were defined as two cells that were connected and within one nuclear length from each other. See Table S2 for antibodies used.

Amersham Cell Proliferation Labeling Reagent (GE Healthcare Life Sciences, Little Chalfont, UK) was administered to quantitate cell proliferation. Mice were given 3 consecutive IP injections of 10ul per gram body weight of 5-bromo-2-deoxyuridine and 5-fluoro-2’-deoxyuridine as recommended by the manufacturer, with the first dose given the day after the last dose of antibody. BrdU immunostaining was performed on paraffin embedded tissue that was processed as described above and incubated at 4°C overnight with a primary anti-BrdU antibody (1:200, GE Healthcare Life Sciences, Little Chalfont, UK), and then with biotinylated anti-mouse IgG secondary (1:500, Vector Laboratories, Burlingame, CA, USA) followed by streptavidin-conjugated Alexa Fluor 594 (1:100, Molecular Probes, Eugene, OR, USA). FITC 488 conjugated anti-GFP antibody, which binds to all four of the confetti variants (Rockland Immunochemicals for Research, Gilbertsville, PA, USA) was used to detect all four confetti reporters at the 488nm wavelength channel (GFP).