Click it plus tunel assay for in

Tissue sections (10–16 micron thickness) from focal KA-injected brain were used for TUNEL assay. Click-iT Plus TUNEL Assay for in situ Apoptosis Detection with Alexa Fluor 488 dye (Thermo Fisher, C10617) kit was used following the manufacturer instruction.

In the frozen sections of the gastrocnemius muscle at 21 days postdenervation sliced to a thickness of 10 μm, the TUNEL staining was performed using the Click-iT™ Plus TUNEL Assay for In Situ Apoptosis Detection, Alexa Fluor™ 594 dye (Thermo Fisher Scientific). Thereafter, to determine the location of the nuclei, the sections stained with the laminin were made by incubations of the anti-laminin IgG (H + L) antibody at 4 °C overnight and then the fluorescent-labeled secondary antibody (Alexa Flour 488-labeled goat anti-rabbit IgG (H + L) at room temperature for 1 h, as described above. Finally, the slides were cover-slipped with ProLong™ Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific) for detection of all the nuclei in each section. For quantification of the TUNEL-positive nuclei, the cross-sectional areas of the specimens were evaluated in ≥ 6 visual fields at 200–400 × magnification per animal. The numbers of each TUNEL- and DAPI-positive nuclei were separately counted in the observed sections and a total of > 400 nuclei in ≥ 6 fields of view were counted for each section. The ratio of TUNEL-positive nuclear to all the nuclei (TUNEL- or DAPI-positive nuclei) was calculated.