Apoptag plus peroxidase in situ apoptosis detection kit

After 7-day post-transplantation to the injured NOD/SCID mice liver, donor hUCMSCs proliferation was quantified by immunohistochemical staining of PCNA. Fresh liver tissues were embedded with OCT medium and “snap-frozen” in dry ice. Frozen sections of 10-μm thickness were prepared and subjected to permeabilization in acetone at –20 °C for 10 min. To reduce non-specific signal, slides were incubated with goat serum blocking buffer (Boster, Wuhan, China) at room temperature for 1-hour. Subsequently, the slides were incubated with primary antibodies PCNA (1:100, Cell Signaling). After washing thrice with PBS, slides were incubated with mouse antibody against mouse IgG conjugated with Alexa flour (1:1000, Cell Signaling). Sections were co-stained with human cytokeratin-18 (hCK-18; 1:100, Abcam HK, NT, HK) and goat antibody against rabbit IgG conjugated with FITC (1:1000, Abcam HK). Apoptosis was quantified by terminal dUPT nick end-labeling (TUNEL) using ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon, Billerica, MA) after 3-day post-transplantation. The number of PCNA cells or apoptotic cells was quantified in 3 microscopic fields at ×40 magnification using ImageJ software.

Terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) was performed under the instruction of the ApopTag plus peroxidase in situ Apoptosis Detection Kit (Chemicon International, USA). Briefly, deparaffinized tissue sections (top part of left testes) were incubated with proteinase K (20 μg/ml), subjected to 3% H₂O₂ at 37 ˚C for 30 min to inhibit endogenous peroxidase, and then incubated with equilibration buffer at room temperature for 1 min. Each section was incubated with TdT (terminal deoxynucleotidyl transferase) at 37 ˚C for 1 h and then washed in stop/wash buffer for 10 min. The sections were incubated in anti-Digoxigenin Peroxidase Conjugate at room temperature for 30 min and were stained with diaminobenzidine (DAB) as a peroxidase substrate. After counterstaining with methyl green, numbers of TUNEL-positive cells per tubule were counted in 50 tubules per animal with the aid of a light microscope. All counting procedures were performed ‘blindly’.

Cell apoptosis was assessed using TUENL, a method that is based on the specific binding O-TdT to the 3-OH ends of DNA, ensuring the synthesis of a polydeoxynucleotide polymer. For this purpose, ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon) was employed according to the recommendation. Omission of the working strength TdT enzyme was considered as a negative control.