Aec substrate

The 6 μm thick consecutive cryostat sections were mounted on uncovered glass slides, air-dried for at least 1 h at room temperature and stored vacuum-sealed at −20 °C. For the staining procedure, the sections were dried again for 30 min at room temperature, fixed in acetone (Merck, Darmstadt, Germany) for 10 min and air-dried again for 10 min. Then the slides were washed in phosphate-buffered saline (PBS) and incubated with 4% bovine serum albumin in PBS (Biomol, Hamburg, Germany) for 20 min followed by incubation with the primary antibody for 45 min at room temperature.
Primary antibodies were diluted at an appropriate ratio in PBS + 1% bovine serum albumin/PBS. Primary antibodies were detected with EnVision conjugated with peroxidase (DakoCytomation, Hamburg, Germany) for 30 min. The substrate reaction for peroxidase was performed with the AEC substrate (DakoCytomation) according to the manufacturer’s instructions. Subsequently, the sections were washed in water, counterstained in 50% hemalum (Merck, Darmstadt, Germany) and mounted with glycerol-gelatin (Merck). The same protocol was performed for negative controls, in which an isotype-matched control antibody was used, which revealed either no staining or only weak background staining. Ki-67 antibody was bought from IMMUNOTECH SAS/Beckman Coulter, Marseille, France.

Liver tissues of fetal mice were fixed with 4% PFA at 4°C for overnight, processed, and embedded in paraffin. Sections (5 µm) were placed on MAS-coated slides for standard histological staining with hematoxylin/eosin (HE). For Cdkn1a staining, antigen retrieval was performed in heated Tris-EDTA buffer (pH 9) with microwave for 15 min. The blocked samples with 10% normal goat serum were incubated with anti-Cdkn1a antibody (1∶25) for overnight at 4°C. As secondary antibody, EnVision⁺ kit (DAKO) was used, and signals were visualized with AEC⁺ substrate (DAKO), according to the manufacturers instruction.

The in situ nick-translation technique (ISNT) was used to staining DNA fragmentation and apoptotic bodies on cell culture slides [20 (link)]. Slides were incubated with proteinase K (20 μg/ml, Qiagen, Germany) for 15 min at room temperature. After rinsing with distilled water the endogenous peroxidase was quenched with 0.3 % hydrogen peroxide for 10 min. Being rinsed once more, the slideswere then equilibrated in nick buffer (Tris, MgCl₂, ß-Mercaptoethanol, 20 mg/ml BSA, distilled water) at room temperature for 10 min. By incubating the slides with dNTPs and biotinylated 7-dATP (Gibco, USA) diluted in nick buffer for 65 min at 37 °C, the in situ nick-translation was performed. Terminating buffer (0.3 mol/L sodium chloride and 0.03 mol/L sodium citrate) was used to rinse the chamber slides at room temperature for 15 min. After having washed the slides in PBS, they were incubated with extravidin–peroxidase (Sigma, Germany) at room temperature for 30 min. AEC-substrate (Dako, Denmark) was used for colour development. Afterwards the slides were counterstained with haemalaun, then washed and mounted. The specificity of ISNT reactivity was confirmed by human epidermis and lymph node sections. 10 replicates were performed. Negative controls were performed by incubation in nick buffer without dNTPs and biotinylated 7-dATP.

Antibodies were obtained as follows: anti-βArr1/2 (D24H9), anti-βArr2 (C16D9), anti-human LDHA (3582), anti-phospho-Src (Y416) (D49G4), anti-Src (36D10), anti-Cyclin D1 (92G2), anti-GAPDH (2118S), and Signal Stain Boost IHC detection reagent from Cell Signaling; anti-human Ki67 (ab92742); anti-Actin (ab3280) from Abcam; anti-HSP90 (610419) from Fischer; anti-Cyclin A (H-432) from Santa Cruz Biotechnology; anti-Flag M2 (F3165) from Sigma; and HRP-coupled anti-rabbit (711-035-152) or anti-mouse (715-035-150) from Jackson Immuno Research Laboratories. Chemical and other reagents were obtained as follows: PP2 (Src family kinase inhibitor) from Selleckchem, protease inhibitor cocktail and puromycin from Sigma-Aldrich; polybrene from Millipore; collagen from Roche; matrigel from BD; Super Signal West Pico chemiluminescent substrate from Thermo Scientific; and Target Retrieval Solution, Protein Block, AEC substrate and Faramount aqueous mounting medium from Dako. High pure RNA isolation kit was from Roche, and iScript™ reverse transcription supermix for RT-qPCR and iQ SYBR green supermix were from Bio-Rad. Control and targeted siRNAs were from Dharmacon (SMARTpool: ON-TARGET plus ARRB2 siRNA) and shRNA bacterial glycerol stock clone ID: NM_004313.3-309s21c1 targeting ARRB2 was from Sigma.