Tunel enzyme

Slides were deparaffinized and rehydrated as above. They were then permeabilized for 8 min in 0.1% sodium citrate containing 0.1% Triton X-100. Slides were afterwards placed in a plastic jar containing 200 mL of 0.1 M citrate buffer pH 6.0 and irradiated at 750 W in a microwave for 1 min for antigen retrieval, and immediately cooled by adding room temperature ddH₂O.
Slides were immersed in Tris-HCl 0.1 M pH 7.5 containing 3% BSA and 20% fetal bovine serum for 30 min at room temperature for blocking and then washed with PBS. Slides were incubated for 1 h at 37 °C with a TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) reaction mixture (11767291910 and 11767305001, TUNEL Label Mix and TUNEL Enzyme, Roche), following the manufacturer’s instructions. Slides were counterstained with Hoechst 33342 and mounted in Prolong™ Gold Antifade Mountant. An amount of 40× images were taken with the Zeiss LSM 700 Confocal microscope, and TUNEL-positive cells in three different retinal sections were manually counted in a blind manner using ImageJ software. Three distinct whole retinal sections per mouse were analyzed and averaged, and at least four animals per group were used.

Cell death was detected by TUNEL using the In Situ Cell Death Detection Kit, Fluorescein (Roche, Basel, Switzerland). Briefly, slides were baked for 1 h following by dewaxing and rehydration. Antigen retrieval was achieved via proteinase K addition (20 µg/mL; Carlsbad, CA, USA). Slides were blocked in 4% goat serum, then incubated overnight with the monoclonal Anti-α-Actinin (Sarcomeric) antibody (A7811, 1:100)(Millipore Sigma), before the addition of TUNEL enzyme (1:10, Roche), goat anti-mouse IgG H&L (Alexa Fluor^® 568) (1:400, ab175473, Abcam) and wheat germ agglutinin, Alexa Fluor™ 647 Conjugate (1:250; Thermo Fisher Scientific). Cell death was imaged as above, and quantified as the number of cardiomyocytes (identified via sarcomeric α-actinin stain) and non-cardiomyocytes normalized to total nuclear number (DAPI).

Cells were fixed with freshly prepared 2% paraformaldehyde for 60 min at room temperature, washed with PBS, and permeabilized with 0.2% Triton X-100 for 2 min at 4 °C. The cells were then incubated with TUNEL reaction mixture containing TUNEL Enzyme and TUNEL label mix (Roche) according to the manufacturer's instructions. Positive staining in the nucleus was identified using FV10i-LIV confocal microscope (Olympus).

Granta‐519 cell line (from DMSZ) was maintained in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum. OCI‐Ly3 and 10 cell lines (DSMZ) were cultured in Iscove's modified Dulbecco's medium (IMDM, Invitrogen) supplemented with 20% human serum and 50 μmol/l 2‐βmercaptoethanol.
For cell death assays, 0.5 × 10⁶/ml of Granta‐519 or 0.1 × 10⁶/ml of OCI‐Ly3/10 cells were starved, respectively, in DMEM or in IMDM 0%SVF and treated with netrin‐1‐interfering net‐1 mAb antibody or an IgG1‐type control antibody at 10 μg/ml in each assay. Netrin‐1 was used at 150 ng/ml in all experiments. Cell counting was performed between 24 and 96 h with a NucleoCounter NC‐3000 (Beckman). Apoptosis was monitored 24–48 h after treatment using the Caspase 3/CPP32 Fluorimetric Assay Kit (Gentaur Biovision). For detection of DNA fragmentation, treated cells were cytospun and TUNEL assay was performed with 300 U/ml TUNEL enzyme and 6 μM biotinylated dUTP (Roche) as previously described (Castets et al, 2009). Human FLAG‐tagged netrin‐1 and net‐1 mAb were obtained from Adipogen (AG‐40B‐0040).
SUDHL4 cell line was cultured in RPMI‐Glutamax medium (Invitrogen) supplemented with 10% fetal bovine serum. After transfection, cells were splitted at 1.25 × 10⁶/ml in serum‐free medium. Cell counting was performed at 120 hours as described above.

TUNEL assay: Detection of DNA fragmentation, a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay was performed by following the protocol of the TUNEL assay kit (Roche). Briefly, fixed cells or tissue samples were permeabilized with 0.2% TX-100 in PBS (30 min at room temperature), washed with PBS, incubated with 300 U ml^−l TUNEL enzyme and 6 μmol l⁻¹ biotinylated deoxyuridine triphosphate (Roche Diagnostics, Mannheim, Germany). The extremities of the biotin coupled DNA were revealed by using Cy-3-coupled streptavidin (1:1,000 in PBS, Jackson Immunoresearch). The slides were washed with PBS, DAPI-stained, then washed with PBS and finally, mounted with Fluoromount G (SouthernBiothec). Images were acquired with Zeiss Axiovision fluorescence microscopy and NIS element AR 4.20.01 Nikon fluorescence microscopy. Caspase-3 activity assay: Cells were first harvested by scraping. Cell pellets were obtained by centrifugation at 4 °C and lysed. The caspase 3 activity assay was performed according to the manufacturer’s instructions (Biovision caspase-3 colorimetric assay kit). Total protein concentrations were measured with the BCA assay kit using BSA as a standard (Pierce Biotechnology, Rockford, IL, USA). Absorbance readings were done on a TECAN infinite F500.

To perform the double staining of Anti-FoxP3- (mouse IgG1, monoclonal 236A/E7, 1:50; Thermofisher, Waltham, MA, USA) and TUNEL staining, another protocol was necessary. The procedure resembles immunohistochemical FoxP3 staining, although the endogenous peroxidase was not blocked. After heat pretreatment, unspecific binding sites and staining were blocked by incubation with Ultra-Vision-Protein-Block (Thermofisher, Waltham, MA, USA) for 15 min. The sections were then incubated with the primary antibody FoxP3 for 16 h at 4 °C. After washing with PBS, the secondary antibody goat-anti-mouse-IgG Cy3-labeled (Jackson Immunoresearch Laboratories, West Grove, PA, USA) was applied for 30 min at room temperature. In the next step, the TUNEL staining was performed. TUNEL enzyme (Roche, Basel, Switzerland) and TUNEL label (Roche, Basel, Switzerland) were mixed in a ratio of 1:10 and 50 µL were applied on each slide. Covered with a cover glass, the sections were incubated for one hour at 37 °C. After incubation and washing in PBS, the sections were air-dried and covered with mounting medium for fluorescence with DAPI (Vector, Burlingame, CA, USA).

In situ TUNEL assay was carried out in ovarian sections using TUNEL enzyme (#11 767 305 001, Roche, Mississauga, ON, Canada) and fluorescent TUNEL label (#11 767 291 910, Roche, Mississauga, ON, Canada) as per the manufacturer’s instructions. Images were obtained (×20 objective) on a Zeiss® Axioplan 2 Imaging microscope, using Axiovision® Release 4.8.2 imaging software. Follicles with visible oocytes were categorized into preantral, early antral, late antral and preovulatory stages, and TUNEL positivity was determined if ≥50% of the granulosa cells had positive staining. TUNEL positivity was expressed per follicle stage as the mean number of TUNEL positive follicles over total follicles, with the mean of three representative slides per rat.