Texas red or fitc conjugated secondary antibodies

Fetal gonads were dissected in phosphate buffered saline (PBS), fixed in 4% paraformaldehyde overnight at 4°C, embedded in paraffin and sectioned. Slides were incubated with anti-GCNA IgM (courtesy of G. Enders, undiluted supernatant), anti-STRA8 (Abcam. 1∶100), and anti-phosphoH2A.X (Upstate Cell Signaling Solutions, 1∶250 dilution). Colorimetric staining was performed using ABC reagents (Vector Laboratories) and developed with DAB peroxidase substrate (Vector Laboratories).
Sections were mounted in Vectashield Medium with DAPI (Vector Laboratories), and fluorescent staining was obtained using Texas-Red or FITC-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, 1∶500 dilution).

To examine the staining pattern of various target proteins in prostate cancer cells, the fixed preparations were first permeablized in 0.5% Triton X-100 for 30 min, blocked with 10% normal goat serum at room temperature for 30 min, and then incubated with SIRT3(Cell Signaling), p-Akt (Ser473) (Cell Signaling), c-MYC (Epitomics) overnight at 4°C. For immunofluorescence analyses, Texas Red- or FITC-conjugated secondary antibodies (Jackson ImmunoResearch) were then used to reveal the labeling patterns. For immunohistochemistry analyses, HRP-conjugated secondary antibody (Jackson ImmunoResearch) was used. Negative controls were performed by skipping the primary antibody step. The labeled tissue and cells were visualized under a microscope (Leica DFC420C) and images were processed using Adobe Photoshop software.

To prepare frozen sections, testes were fixed in 4% paraformaldehyde (PFA) at 4 °C overnight, embedded and sectioned (6 μm). The sections were treated with 6 μM DTT and 10% serum in TBST (1 × TBS containing 0.1% Tween 20) for 1 h at room temperature (RT), then incubated overnight at 4 °C with primary antibodies diluted in 10% serum in TBST. After incubation with secondary antibodies, the sections were then washed in TBS three times and stained with DAPI (Vector Laboratories). For whole-mount assay, seminiferous tubules from adult testis were prepared as previously described with modifications [54 (link)]. Testis tubules were digested with trypsin and collagenase, washed with PBS, and fixed with 4% PFA for 2 h. After blocking with 10% serum in TBST (1 × TBS containing 0.1% Tween 20) for 1 h at RT, the samples were incubated overnight at 4 °C with anti-MOV10 antibody (1:25) and anti-PLZF antibodies (1:200), washed three times in TBS, and incubated with Texas red or FITC-conjugated secondary antibodies (Jackson Immuno Research) for 1 h at RT. Samples were washed as before and mounted in microslide shield with DAPI. Immunofluorescence for all samples was examined under laser scanning confocal microscope (Carl Zeiss, LSM700).