An Optimized Protocol for Histochemical Detection of Senescence-associated Beta-galactosidase Activity in Cryopreserved Liver Tissue

Jannone, Giulia;Rozzi, M;Najimi, Mustapha;Decottignies, Anabelle;Sokal, Etienne
(2020) Journal of Histochemistry and Cytochemistry : imaging the spectrum of cell biology — Vol. 68, n° 4, p. 269-278 (2020)

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Senescence-associated beta-galactosidase (SA-β-gal) activity assay is commonly used to evaluate the increased beta-galactosidase (β-gal) activity in senescent cells related to enhanced lysosomal activity. Although the optimal pH for β-gal is 4.0, this enzymatic activity has been most commonly investigated at a suboptimal pH by using histochemical reaction on fresh tissue material. In the current study, we optimized a SA-β-gal activity histochemistry protocol that can also be applied on cryopreserved hepatic tissue. This protocol was developed on livers obtained from control rats and after bile duct resection (BDR). A significant increase in β-gal liver activity was observed in BDR rats vs controls after 2 hr of staining at physiological pH 4.0 (6.98 ± 1.19% of stained/total area vs 0.38 ± 0.22; p<0.01) and after overnight staining at pH 5.8 (24.09 ± 6.88 vs 0.12 ± 0.08; p<0.01). Although we noticed that β-gal activity staining decreased with cryopreservation time (from 4 to 12 months of storage at -80C; p<0.05), the enhanced staining observed in BDR compared with controls remained detectable up to 12 months after cryopreservation (p<0.01). In conclusion, we provide an optimized protocol for SA-β-gal activity histochemical detection at physiological pH 4.0 on long-term cryopreserved liver tissue.
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Jannone, G., Rozzi, M., Najimi, M., Decottignies, A., & Sokal, E. (2020). An Optimized Protocol for Histochemical Detection of Senescence-associated Beta-galactosidase Activity in Cryopreserved Liver Tissue. Journal of Histochemistry and Cytochemistry : imaging the spectrum of cell biology, 68(4), 269-278. https://doi.org/10.1369/0022155420913534 (Original work published 2020)