5 bromo 4 chloro 3 indolyl β d galactopyranoside x gal

The generation of the rAd-Tat vector derived from a child with HIVAN has been described in detail previously (Xie et al., 2014 (link)). Briefly, a cDNA fragment encoding the full-length Tat protein was cloned into the pCXN2-FLAG vector and used to generate E1-deleted recombinant adenoviruses carrying Tat-HIVAN-FLAG (Xie et al., 2014 (link)). The protein sequence of the Tat-HIVAN gene was aligned and compared to other Tat genes using the Clustal Omega multiple sequence alignment program (www.ebi.ac.uk/Tools/msa/clustalo/). Both Tat-FLAG and LacZ control adenoviruses were amplified, purified, desalted and titrated as described previously (Kozarsky et al., 1993 (link); Jerebtsova et al., 2005 (link), 2007 (link)). The pfu ratio of the virus stock used in these experiments was 100. To detect the localization of β-galactosidase (LacZ) expression, frozen tissue sections (10 μm) were fixed in 0.5% glutaraldehyde (Sigma-Aldrich) at room temperature for 10 min, washed with PBS and stained for 2 h at 37°C in PBS containing 5 mM K3 Fe(CN)6, 5 mM K4 Fe(CN)6, 1 mM MgCl₂ (all from Sigma-Aldrich), and 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal, Boehringer Mannheim). The sections were then counterstained with hematoxylin (Fisher Scientific) and mounted for microscopic evaluation.

The generation of the rAd-FGF-2 and rAd-LacZ vectors was described in detail in previous studies. Both FGF-2 and LacZ control adenoviruses were amplified, purified, desalted and titrated as described previously (Kozarsky et al., 1993 (link); Jerebtsova et al., 2007 (link)). The rAd-FGF-2 vectors carry a 700 bp cDNA sequence encoding a secreted form of human FGF-2 (rAd-FGF-2) (Gupta et al., 2001 (link)), while the rAd-LacZ vector carries the Escherichia coli LacZ gene (rAd-LacZ) described previously (Kozarsky et al., 1993 (link)). To confirm the expression of β-galactosidase (LacZ) in the liver, frozen tissue sections (10 μm) were fixed in 0.5% glutaraldehyde (Sigma-Aldrich) at room temperature for 10 min, washed with PBS and stained for 2 h at 37°C in PBS containing 5 mM K3 Fe(CN)₆, 5 mM K4 Fe(CN)₆, 1 mM MgCl₂ (all from Sigma-Aldrich) and 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal; Boehringer Mannheim). The sections were then counterstained with Hematoxylin (Thermo Fisher Scientific) and mounted for microscopic evaluation.

Dimri’s staining protocol, was used to detect cellular senescence [83 (link)]. Briefly, cells were seeded in a 3.5 cm dish and cultured for at least 72 h. They were washed in Dulbecco’s phosphate-buffered saline (PBS; Sigma-Aldrich, St. Louis, MO, USA, D8537) for 5 min. To fix the cells, a fixation solution containing 0.2% glutaraldehyde solution (Sigma-Aldrich, G5882) and 2% formaldehyde solution (Merck KGaA, Darmstadt, Germany, 104003) was prepared in PBS. After 5 min of fixation, the cells were washed twice with PBS for 5min. Subsequently, the cells were incubated overnight at 37 °C (without CO₂) in the SA-β-Gal staining solution, which contained 5 mM potassium ferricyanide (III) (KGaA, 104973), 5 mM potassium ferrocyanide (II) (Sigma-Aldrich, St. Louis, MO, USA, P9387), 2 mM MgCl₂ (Sigma-Aldrich, M1028), 150 mM NaCl (Sigma-Aldrich, St. Louis, MO, USA, M1028), 0.5 mg/mL 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal; Roche, Basel, Germany, 3117073001), and 40 mM citrate/sodium phosphate buffer (Sigma-Aldrich, St. Louis, MO, USA, S5136), pH = 6. For the analysis, 1000 cells were counted from each sample with a Axiovert 40 CFL bright field microscope. The senescence test was performed at each passage and before the experiments as well as after seeding in some experiments to ensure that the cultures exhibited the same percentage of senescent cells to allow comparison.