Alexa fluor 488 anti rabbit antibody

Embedded tissues were cut into 5 μm thick and placed in electrocharged slides; then, histological sections were deparaffinized and gradually rehydrated. For histological examination, samples were stained using the standard H&E method. For IF, after sample rehydration, antigen was retrieved according to the Rodent Decloaker kit (BioCare Medical. CA, USA). Then, they were blocked with 1% BSA for 2 h and incubated overnight at 4 °C with the following primary antibodies: Annexin A1 (ANXA1, 1:50; NBP1-90162; RRID: AB_11018791), Annexin A2 (ANXA2, 1:50; NBP1-31310; RRID: AB_2242775), and Annexin A5 (ANXA5, 1:50; NBP2-38248); all antibodies were from NOVUS Biologicals. CO, USA. Then, tissues were incubated for 1 h at room temperature in the dark with an Alexa Fluor 488 anti-rabbit antibody (1:300, ab150077; RRID: AB_2630356, Abcam, MA, USA). Next, nuclear DNA was stained with DAPI (1:1000; 62.247; Thermo Scientific, IL, USA) for 5 min at room temperature. Finally, tissues were fixed with Fluoroshield mounting medium (Thermo Scientific, IL, USA), and IF images were captured with ZEISS Axio-A1 Microscopy (Carl-Zeiss, Oberkochen, Germany).

The soleus muscles samples were cut into 5-μm sections. The slides were deparaffinized and rehydrated subsequently. Primary CD31 antibody (anti-PECAM-1 polyclonal antibody; Abcam, USA) diluted 1:200 were incubated on slides overnight at 4 °C. And the secondary antibody used for visualization was Alexa Fluor 488 anti-rabbit antibody (Abcam, USA). The sections were stained with 4′-6-diamidino-2-phenylindole (DAPI) and mounted, and then observed under fluorenscence microscopy and images were captured with a digital camera. According to the methods described by Hata¹⁶, the number of capillary and muscle fibers was counted blindly in 10 fields from each section by three independent investigators and then the ratios (capillary to muscle fiber) were calculated out.