Immunohistochemistry and tissue samples
Formalin-fixed, paraffin embedded sections of mouse and human organs were cut into 6 μm thin sections, deparaffinised and thereafter rehydrated. Epitope retrieval was then performed in 10 mM Tris-EDTA-buffer pH 9,0 during 4 min in microwave oven 850 W followed by 15 min 150 W. After cooling down for 20 min at RT, samples were rinsed properly in water. Concerning the staining procedure, the slides were firstly blocked for 10 min in 3% BSA in PBS. After rinsing in Tris-HCl pH 7,4, incubation of primary antibody was done for 60 min in 3% BSA/PBS. Following few rinses, appropriate secondary antibody (Dako EnVision anti-rabbit or anti-mouse) incubation was done for 30 min. Slides, again rinsed, were then incubated in DAB+ liquid Dako (K3468) for 10 min, and then rinsed in water. Samples were incubated in Mayers HTX for 1 min, rinsed with tap water, and finally dehydrated, cleared and mounted. Human testicular samples were obtained from patient diagnosed testicular neoplasm and therefore underwent orchiectomy.
Tissues sample homogenenisation and RNA extraction
Liquid nitrogen frozen mouse samples were homogenised using the MagNA Lyser and MagNA Lyser Green Beads. Briefly, RNA and protein samples were homogeneised in respective lysing buffers, RA1 from Macherey Nagel for RNA; RIPA buffer (1X PBS; 1% Nonidet P-40; 0.5% sodium deoxycholate; 0.1% SDS) for protein samples. 1 to 4 cycles (6500 rpm, 50 s) were used to homogeneise the tissues, an ice-cooling step of 2 min being done between each cycle. Total RNA was extracted and cleaned up from the lysate using the Nucleospin kit (Macherey Nagel), including a DNAse treatment step.
The following antibodies were used for immunohistochemical stainings of paraffin embedded tissues: CIP2A: rabbit polyclonal anti-CIP2A, MYC: mouse monoclonal 26 9E10 (sc-40, Santa Cruz Biotechnology) and S62-MYC: rabbit polyclonal anti-S62-MYC.
For cDNA synthesis 1 μg total RNA was incubated with 250 ng random hexamer for 5 min at 70°C, then cooled down on ice for another 5 min. Total RNA was reverse transcribed in a final volume of 25 μL containing enzyme buffer, 10 units of RNAse inhibitor, DTT, 0,5 mM deoxynucleotide triphosphate, and 5 units MMLV reverse transcriptase. The samples were incubated at room temperature for 10 min, then at 42°C for 50 min. The reverse transcriptase was finally inactivated by heating at 70°C for 15 min before PCR amplification. The quantification was based on the standard curve method. The data were normalised using β-actin. Oligonucleotides were obtained from Proligo. For quantitative real-time PCR, 2 μL of diluted reverse transcription reaction samples (1/10) were added to 8 μL of a PCR mixture made up of 5 μL of PCR Master Mix (Applied Biosystems), 1 μL of each primer at a concentration of 3 μM, and 1 μL of specific probe at a concentration of 2 μM. The thermal cycling conditions comprised an initial step at 50°C for 2 min and a denaturation step at 95°C for 10 min followed by 40 cycles at 95°C for 15 and 60°C for 1 min. All PCRs were carried out using an ABI Prism 7000 Sequence Detection System (Applied Biosystems). The specificity of each primer couple was shown by a dissociation curve analysis. Results are derived from the average of at least two independent experiments. Gene expression was reported relative to housekeeping gene.
Surgically induced cryptorchidism and Busulfan injection
Surgical unilateral cryptorchidism (Crypt) of the right testes was induced in nine male mice (C57BL/6) at 8 weeks of age. Each mouse was anaesthetised, and a lateral incision was made on the abdominal wall above the inguinal canal. The right testis was pulled away from the scrotum into the abdominal cavity. The inguinal canal was closed with nonabsorbable sutures. The mice were sacrificed after 6 months, and both testes were collected for histological and quantitative polymerase chain reaction (PCR) (qPCR) analyses.
Busulfan (Busilvex, Myleran, GlaxoSmithKline, UK) was dissolved in dimethyl sulphoxide and mixed with an equal volume of sterile distilled water before intraperitoneal injection into 8 week old C57BL/6 mice. Mice were treated with 20 or 30 mg/kg busulfan. Control mice were injected with 30 mg/kg dimethyl sulphoxide. 4 weeks after injection the mice were sacrificed and testes were collected for histological and quantitative polymerase chain reaction (PCR) (qPCR) analyses.