Senescence-associated β-galactosidase assay

Dear Luisa,

From our own experience, we acknowledge that the SA-b-Gal assay contains critical steps that must be followed to ensure a proper staining of senescent cells. We are currently writing a Bio-Protocol article in which we detail the procedures to perform SA-b-Gal staining on adult zebrafish. In the meantime, here is some advice to improve the quality of the assay:

1 - Fish fixation step: PFA quality is essential to guarantee proper fish fixation. We improved the quality of our results by routinely preparing fresh 4% PFA in PBS using a commercial 16% PFA (Thermo Scientific™, catalog number: 28908). To ascertain a proper fixation, we incubate fish in 4% PFA for 3 days at 4°C on a roller mixer.

2 - SA-b-Gal staining: Incubating fish at pH 6.0 is crucial to guarantee an optimal senescence-dependant b-galactose reaction.

3 - Decalcification step: incubation with EDTA 0.5M pH 8.0 should not exceed 48h. Samples should then be washed extensively with water for at least 30min to fully remove EDTA prior to the embedding procedure.

4 - Embedding procedure program:

1. 10% buffered formalin for 1h
2. Distilled water for 2 min
3. EtOH 70% for 2h
4. EtOH 95% for 1h
5. EtOH 100% for 1h
6. EtOH 100% for 1h
7. EtOH 100% for 2h
8. EtOH 100% for 1h
9. Xylene for 30min
10. Xylene for 1h
11. Xylene for 1h
12. Paraffin for 30min at 62°C
13. Paraffin for 1h at 62°C
14. Paraffin for 1h at 62°C

We hope these clarifications will help you to improve the efficiency of this assay.

Best regards,

The authors

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