Can the Senescence β-Galactosidase Staining Kit #9860 be used with frozen or paraffin-embedded (FFPE) tissues?

CST has not tested the Senescence β-Galactosidase Staining Kit #9860 successfully on tissue sections (frozen or paraffin-embedded), so we do not have a tissue-specific protocol to provide.

Attempts to use this kit on paraffin-embedded tissue samples have not yielded satisfactory results. The kit is designed for detecting β-galactosidase activity at pH 6, which is a characteristic of senescent cells​. It has been reported that paraffin-embedding inactivates the​ enzymatic activity of β-galactosidase. All successful X-gal staining procedures found in the literature involve fresh-frozen tissues, whole mounts, or cryosections of fixed tissue, not formalin-fixed, paraffin-embedded (FFPE) tissues. Therefore, it is highly unlikely that this kit will work ​with ​F​FPE samples.

We are aware of many customers who use this kit on frozen tissue samples, and there are several publications that demonstrate the successful use of this kit under these conditions. A few are listed below for reference: 

• Idelfonso-García OG, Pacheco-Rivera R, Alarcón-Sánchez BR, et al. Protocol to detect senescence-associated β-galactosidase and immunoperoxidase activity in fresh-frozen murine tissues. STAR Protoc. 2024;5(2):103009. doi.org/10.1016/j.xpro.2024.103009.
• Raffaele M, Kovacovicova K, Bonomini F, Rezzani R, Frohlich J, Vinciguerra M. Senescence-like phenotype in post-mitotic cells of mice entering middle age. Aging (Albany NY). 2020;12(14):13979-13990. doi.org/10.18632/aging.103637
• Childs BG, Bussian TJ, Baker DJ. Cellular Identification and Quantification of Senescence-Associated β-Galactosidase Activity In Vivo. Methods Mol Biol. 2019;1896:31-38. doi: 10.1007/978-1-4939-8931-7_4

To the best of our knowledge, customers working with frozen tissue follow our protocol closely. One recommendation we have is to use a chamber slide or glass slide with a silicon gasket, which will allow you to fully submerge your tissue sections and prevent evaporation. The amount of staining solution you use will depend on the chamber size you have. It is important to keep your samples fully submerged when incubating overnight at +37°C to minimize evaporation. We also recommend using a positive control to ensure that the staining solution is performing as expected. We frequently use NIH/3T3 cells treated with 12.5 μM etoposide for 24 hours and allowed to recover for 3 to 4 days. This will induce cellular senescence and give a positive blue staining result.
 

Last updated: April 23, 2026