Click it plus tunel assay for in situ apoptosis detection kit

TUNEL staining of frozen tissue slides was performed using the Click-iT Plus TUNEL Assay for In Situ Apoptosis Detection kit (Thermo Fisher Scientific, C10619). Briefly, solutions outlined by the kit were prepared before starting the staining procedure. Frozen tissue sections were brought to room temperature in PBS for 15 min, and the tissue was encircled with a PAP pen. Tissues were fixed in 4% formaldehyde in PBS at 37 °C, washed twice for 5 min in PBS, and subsequently incubated for 15 min with proteinase K solution at room temperature. Next, slides were washed in PBS for 5 min, fixed again in 4% formaldehyde in PBS for 5 min at 37 °C, and rinsed in deionized H2O. Incubation of slides with 100 μL of compound A for 10 min at 37 °C followed, after which the tissue was incubated with 50 μL of TdT reaction mixture for 60 min at 37 °C. After a deionized H2O rinse, slides were washed in 0.2 μm filtered 3% bovine serum albumin 0.1% PBST for 5 min, rinsed once more in PBS, and incubated with 50 μL of reaction cocktail for 30 min at 37 °C. Tissue slides were rinsed in PBS and stained with DAPI at 1:750 for 5 min. Slides were mounted as described above.

Apoptosis was assessed in WT, p75^NTR−/− and p75^NTRCys259 CGNs treated for 24 h with either 500 μM or 1000 μM AraC starting at 4 DIV. Apoptotic cells were labelled using Click-iT plus TUNEL assay for in situ apoptosis detection kit (Thermo Scientific, Cat: C10617) according to manufacturer instructions. Neurons were also stained for cleaved caspase 3 (Cell Signalling Technology, 9761, 1:400), β-III tubulin and DAPI (Sigma; D9542; 1:10,000) following the protocol for immunocytochemistry explained below. For each experiment and treatment, neurons were cultured in duplicates, and at least 15 images were taken per coverslip with a Zeiss Axioplan confocal microscope. The number of cells positive for cleaved caspase 3 and TUNEL was quantified using NIH ImageJ software.

TUNEL staining of frozen tissue slides was performed using the Click-iT Plus TUNEL Assay for In Situ Apoptosis Detection kit (Thermo Fisher Scientific, C10619). Briefly, solutions outlined by the kit were prepared before starting the staining procedure. Frozen tissue sections were brought to room temperature in PBS for 15 min, and the tissue was encircled with a PAP pen. Tissues were fixed in 4% formaldehyde in PBS at 37° C, washed twice for 5 min in PBS, and subsequently incubated for 15 min with proteinase K solution at room temperature. Next, slides were washed in PBS for 5 min, fixed again in 4% formaldehyde in PBS for 5 min at 37° C, and rinsed in deionized H2O. Incubation of slides with 100 μL of compound A for 10 min at 37°C followed, after which the tissue was incubated with 50 μL of TdT reaction mixture for 60 min at 37° C. After a deionized H2O rinse, slides were washed in 0.2 μm filtered 3% bovine serum albumin 0.1% PBST for 5 min, rinsed once more in PBS, and incubated with 50 μL of reaction cocktail for 30 min at 37° C. Tissue slides were rinsed in PBS and stained with DAPI at 1:750 for 5 min. Slides were mounted as described above.

Specimens were harvested, fixed, decalcified, and sectioned as described for immunofluorescence assays. Apoptotic cells were detected using Click-it Plus TUNEL Assay for In Situ Apoptosis Detection kit (Thermo Fisher, C10617) following the manufacturer’s recommended protocol.

Heart sections prepared and processed as described above. Apoptosis assay was performed by using the Click-iT Plus TUNEL Assay kit for In situ Apoptosis Detection (Thermo Fisher Scientific) as per the manufacturers' protocol that is based on the principle of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL).