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:

衰老相关的β-半乳糖苷酶（SA-β-gal）活性测定通常用于评估与溶酶体活性增强相关的衰老细胞中增加的β-半乳糖苷酶（β-gal）活性。尽管β-gal的最佳pH为4.0，但最常见的是在次最佳pH下通过对新鲜组织材料进行组织化学反应来研究这种酶活性。在当前的研究中，我们优化了SA-β-gal活性组织化学方案，该方案也可应用于冷冻保存的肝组织。此协议是针对从对照大鼠和胆管切除术（BDR）获得的肝脏制定的。在生理pH 4.0染色2小时后，与对照组相比，BDR大鼠的β-gal肝活性显着增加（染色/总面积的6.98±1.19％vs 0.38±0.22；p<0.01）和在pH 5.8下过夜染色后（24.09±6.88对0.12±0.08; p <0.01）。尽管我们注意到，β-gal活性染色随冷冻保存时间的延长而降低（在-80°C下保存4到12个月；p <0.05），但在冷冻保存后长达12个月，与对照相比，BDR中观察到的染色增强仍可检测到（p < 0.01）。总之，我们为长期低温保存的肝组织在生理pH 4.0下的SA-β-gal活性组织化学检测提供了优化的方案：