Can the Senescence β-Galactosidase Staining Kit #9860 be used with frozen or paraffin embedded 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, however, aware of many customers who use this kit on frozen tissue samples and there are several publications that have successfully used this kit under these conditions. We have listed a few of them below for reference.

B. G. Childs, T. J. Bussian, D. J. Baker (2019) Cellular Identification and Quantification of Senescence-Associated Beta-Galactosidase Activity in Vivo Methods Mol Biol. 1896: 31-38.
Baker, D. J. et al. (2004) BubR1 insufficiency causes early onset of aging-associated phenotypes and infertility in mice. Nat. Genetics 36, 744-749.
Swarbrick, A. et al. (2008) Id1 cooperates with oncogenic Ras to induce metastatic mammary carcinoma by subversion of the cellular senescence response. Proc Natl Acad Sci U S A 105, 5402-7.

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 which will allow you to fully submerge your tissue sections and prevent evaporation. You may find examples of chamber slides here. The amount of staining solution you use will be determined by the chamber size you have. It is important to keep your samples fully submerged when you incubate overnight at +37C and to prevent as much evaporation as possible. We would also recommend using a positive control to ensure that the staining solution is performing as expected. We frequently use NIH/3T3 cells treated with 12.5uM etoposide for 24 hours and allowed to recover for 3-4 days. This will induce cellular senescence and give a positive blue staining result.
 

Last updated: October 16, 2024