Sa β gal

Ten micrometres of frozen sections of OCT-embedded tumours were stained for the senescence marker SA β-Gal according to the manufacturer's instructions (Cell Signaling) for B16F10 tumours the staining was performed with dodecanoylaminofluorescein di-β-D-galactopyranoside (C12FDG; Life Technologies) at 33 μM during 2 h at 37 °C at pH 6.0.

The cellular senescence was determined with senescence associated-β-galactosidase (SA-β-gal) staining, which was performed using a senescence associated-β-galactosidase staining kit (Cell Signaling Technology) in accordance with the manufacturer’s guidelines. The blue stained cells were evaluated from 10 different fields. The results were presented as a percentage of positive cells out of total cells.

HIV‐1 Tg and control non‐Tg rats at 6 weeks of age, and rats of meth treatment were sacrificed, and brains were fixed in 4% paraformaldehyde overnight. Brain tissues were sectioned at 40 μm thickness. Brain sections were washed extensively in 1xPBS and then incubated in the sodium citrate buffer (pH 6.0) at 45 °C for 2 h. SA‐β‐Gal staining of these tissues was performed according to the manufacture's protocol (Cell Signaling).

A cytochemical assay that uses the chromogenic compound X-gal (5-bromo-4-chloro-3-indoyl β-D-galactopyranoside) as substrate for the pH-dependent β-galactosidase (SA-βGAL) was used to determine senescence in fixed cells, according to the manufacturer’s instructions (Cell Signaling). The cleavage of X-Gal by β-galactosidase at pH 6 renders an insoluble intracellular blue precipitate that allows the visual discrimination of senescent cells. The percentage of stained cells, as seen by bright field microscopy, was determined with respect to the total number of cells, as revealed by fluorescent nuclear staining using DAPI. In some experiments, cells were immunostained with anti-p75^NGFR antibodies to discriminate human SCs from fibroblasts.

SA-β-gal staining was determined following the manufacturer’s instructions (Cell Signaling Technology) and the steps shown in our previous study [14 (link)]. The senescent cells were detected as blue precipitated cells in the cytoplasm with magnification of x200 [8 (link)]. SA-β-gal positive cells numbers were determined in three fields using Image J software as previously described in a recent study [14 (link)].

For studies on cell expansion, chondrocytes from 3‐week‐old Col2a1‐Cre; loxP‐stop‐loxP ZsGreen mice were digested overnight using 0.4 mg/ml Collagenase P. Cells sorted as zsGreen‐positive chondrocytes were plated at 10,000 cells/cm² and passaged weekly. In the final passage, some wells were stained for SA β‐gal (Cell Signaling Technologies, Danvers, MA) according to the manufacturer's recommendations. After DAPI counterstain, five matched bright field and fluorescent images were taken for each independent cell preparation to quantify the percentage of positively stained cells.