Whole-mount Senescence-Associated Beta-Galactosidase (SA-β-GAL) Activity Detection Protocol for Adult Zebrafish

Materials and reagents

Animals

Adult zebrafish (Danio rerio) 9-month-old

Materials

1. Polypropylene centrifuge tube 15 mL (Corning, catalog number: 352096)

2. Glass microscope Superfrost plus slides 25 × 75 × 1 mm (Menzel Gläser, catalog number: J1800AMNZ)

3. Cover Glass 24 × 36 mm (Labelians, catalog number: LCO2436)

   
   

Reagents

1. Tricaine/MS-222 (Pharmaq)

2. Tris base (Sigma-Aldrich, catalog number: T-1503)

3. PFA 16% (Thermo Scientific^TM, catalog number: 28908)

4. Potassium hexacyanoferrate(II) trihydrate (Sigma-Aldrich, catalog number: P9387)

5. Potassium hexacyanoferrate(III) (Sigma-Aldrich, catalog number: 244023)

6. X-Gal (5-Bromo-4-Chloro-3-Indolyl-B-D-Galactopyranoside) (Sigma-Aldrich, catalog number: B4252)

7. N,N-Diméthylformamide (DMF) (Sigma-Aldrich, catalog number: 227056)

8. Magnesium Chloride Hexahydrate (MgCl₂·6H₂O, Sigma-Aldrich, catalog number: 7791-18-6)

9. Sodium Chloride (NaCl, Sigma-Aldrich, catalog number: S7653)

10. Potassium Chloride (KCl, Sigma-Aldrich, catalog number: 7447-40-7)

11. Sodium phosphate dibasic (Na₂HPO₄, Sigma-Aldrich, catalog number: 71505)

12. Potassium phosphate dibasic (KH₂PO₄, Sigma-Aldrich, catalog number: 1.05104)

13. Ethylenediaminetetraacetic acid (EDTA, VWR, catalog number: A10713.0I)

14. Nuclear Fast red (Sigma-Aldrich, catalog number: 1001210500)

15. Histology cassettes (Simport, catalog number: M505)

16. Embedding Molds (Sakura Finetek, catalog number: 4133)

17. 10% buffered Formalin (VWR, catalog number: 9713.9010)

18. Xylene (VWR, catalog number: 28973.363)

19. Ethanol 100% (EtOH, VWR, catalog number: VWRC20821.330)

20. Paraffin (Merck Millipore, catalog number: 1.11609.9025)

21. 1 M Tris pH 9 (see Recipes)

22. Tricaine/MS-222 400 mg/L solution (see Recipes)

23. Tricaine/MS-222 200 mg/L solution (see Recipes)

24. 4% Paraformaldehyde (see Recipes)

25. 10× Phosphate Buffered Saline (PBS, pH 7.4) (see Recipes)

26. 1× Phosphate Buffered Saline (PBS, pH 7.4) (see Recipes)

27. 1× Phosphate Buffered Saline (PBS, pH 6) (see Recipes)

28. Ethylenediaminetetraacetic acid (EDTA, 0.5 M, pH 8) (see Recipes)

29. X-Gal stock solution (25 mg/mL) (see Recipes)

30. X-Gal staining solution (see Recipes)

31. Potassium hexacyanoferrate (II) trihydrate (0.5 M) (see Recipes)

32. Potassium hexacyanoferrate (III) (0.5 M) (see Recipes)

33. Magnesium Chloride (MgCl₂ 0.5 M) (see Recipes)

34. X-Gal staining solution (see Recipes)

35. Ethanol 70% (see Recipes)

36. Ethanol 95% (see Recipes)

Equipment

1. Roller mixer (CAT Ingenieurbuero, catalog number RM5)

2. Dry incubator at 37°C (Memmert, catalog number: UNB 100)

3. NanoZoomer Digital slide scanner (Hamamatsu, catalog number: C13140-01)

4. Tissue processor (Leica Biosystems, catalog number: HistoCore PEARL)

5. Tissue Embedding Console System (Sakura Finetek, catalog number: Tissue-Tek^® TEC^TM 5)

6. Automated Rotary Microtome (Leica Biosystems, catalog number: RM2255)

Software

1. NDP.view2 Image viewing software (U12388-01 Hamamatsu) to open and analyze NanoZoomer scans. Free download from https://www.hamamatsu.com/eu/en/product/life-science-and-medical-systems/digital-slide-scanner/U12388-01.html

Procedure

1. SA-β-GAL Staining procedure

   Day 0

   Starve the fish overnight for at least 17 h, leaving them in their aquarium.

   
   

   Day 1

   1. Sacrifice fish by placing them in a container with 50 mL of 200 mg/L Tricaine MS-222 for 30 min.

      Note: Death can be determined by checking cessation of breath and gill movements.

   2. Proceed with fixation by individually incubating fish in 15 mL centrifugation tubes containing at least 10 mL of 4% PFA for 3 days, at 4°C on the roller mixer.

      Notes:

      1. Differences in PFA quality might affect fixation of the entire fish and, therefore, the quality of β-galactosidase detection. It is then important to prepare fresh 4% PFA solution for each experiment.

      2. Incubation with gentle agitation helps the penetration of PFA and improves fixation of inner organs.

         
         
   Day 4

   1. Wash each sample three times for 1 h in at least 10 mL of PBS pH 7.4, at 4°C on the roller mixer.

   2. Wash each sample 1 h in at least 10 mL of PBS pH 6.0, at 4°C on the roller mixer.

      Note: Pre-incubating fish at pH 6.0 is crucial to ensure an optimal SA-β-GAL reaction in the next step of the protocol.

   3. Incubate each fish with at least 10 mL of X-Gal staining solution for 24 h at 37°C in the dark.

      Note: It is crucial to ensure that SA-β-GAL activity detection is performed at pH 6.0. Lowering the pH below pH 5.9 results in false positives, while increasing the pH above pH 6.1 produces false negatives.

      
      

   Day 5

   1. Wash each sample three times for 5 min in at least 10 mL of PBS pH 7.4 at room temperature on the roller mixer.

   2. Proceed with de-calcification for 48 h in 10 mL of 0.5 M EDTA pH 8 at room temperature.

      Note: To avoid loss of SA-β-GAL staining, fish should not be incubated for more than 48 h in EDTA.

      
      

2. Embedding procedure

   1. After de-calcification, remove EDTA by incubating fish in water for at least 30 min at room temperature.

   2. Insert each sample into a histology cassette.

   3. Proceed with the embedding of the samples in a tissue processor using the following program:

      1. 10% buffered formalin for 1 h

      2. Distilled water for 2 min

      3. Ethanol 70% for 2 h

      4. Ethanol 95% for 1 h

      5. Ethanol 100% for 1 h

      6. Ethanol 100% for 1 h

      7. Ethanol 100% for 2 h

      8. Ethanol 100% for 1 h

      9. Xylene for 30 min

      10. Xylene for 1 h

      11. Xylene for 1 h

      12. Paraffin for 30 min at 62°C

      13. Paraffin for 1 h at 62°C

      14. Paraffin for 1 h at 62°C

   4. Using the Tissue Embedding Console System, insert each sample into an embedding mold and pour paraffin into the mold. Allow for solidification of the paraffin block by placing the mold containing each sample and paraffin onto the cryomodule plate of the Tissue Embedding Console.

   5. Counterstaining procedure

      1. Prepare 5 µm section slides from each paraffin-embedded sample using a microtome.

      2. Counterstain the slides with Nuclear fast red, following the manufacturer’s protocol. Briefly, after conventional deparaffinization and rehydration, wash slides for 1 min at room temperature in distilled water and stain them by incubating for 10 min at room temperature in Nuclear fast red-aluminum sulfate solution 0.1%. After 1 min, wash in distilled water and proceed with conventional dehydration and xylene incubation prior to slide mounting.

         
         

3. Image acquisition

   1. Acquire images using a bright-field microscope or slide scanner.

      Note: The images of the different tissues shown in Figure 1 have been selected from a scan of a whole-body fish section. The slide scans were acquired with a NanoZoomer Digital slide scanner with a 40× source lens and analyzed with the NDPi software.

      
      

      
      Figure 1. SA-β-GAL staining allows the detection of cell senescence in prematurely aged zebrafish tissues. A) Representative images of fish assayed for SA-β-GAL activity. Blue staining shows the presence of SA-β-GAL in different tissues of prematurely aged fish (tert-/-) compared to age-matched controls (wild type, WT). Corresponding scale bar represented in each panel B) Zoom-in of gut villi in A, allowing to precisely distinguish the cells stained in blue. Scale bar 50 μm.

      Note: A significant increase in SA-β-gal positive cells is seen by 9 months of age in tert-/- tissues and by 24 months in WT tissues.

      
      

      Concluding Note:

      We analyzed different tissues of adult zebrafish for presence of SA-β-GAL blue staining. Intensity and localization may change due to biological or technical issues (i.e., age of the fish, health status, penetration of the fixative, longitudinal sections, etc.). Thus, it is crucial to have the proper number of technical and biological replicates for effective result interpretation. In our experiments, we performed a minimum of three biological replicates for each genotype and age.

   Recipes

   1. 1 M Tris pH 9

      1. Dilute 121.4 g tris base in 850 mL of Milli-Q water.

      2. Adjust pH to 9.

      3. Bring volume to 1 L.

   2. Tricaine/MS-222 400 mg/L solution

      1. Dissolve 2 g of tricaine/MS-222 in 489.5 mL of Milli-Q water.

      2. Add 10.5 mL of 1 M Tris (pH 9).

      3. Adjust pH to 7.

   3. Tricaine/MS-222 200 mg/L solution

      Add 50 mL of tricaine/MS-222 to 50 mL of fish system water.

   4. 4% Paraformaldehyde

      Make fresh solution by adding 10 mL PFA 16% to 30 mL of PBS.

   5. 10× Phosphate Buffered Saline (PBS, pH 7.4)

      1. Add 80 g of NaCl, 2.0 g of KCl, 14.4g of Na₂HPO₄, and 2.4 g of KH₂PO₄ to 800 mL of Milli-Q water.

      2. Adjust pH to 7.4.

      3. Bring volume to 1 L.

      4. Autoclave and store at room temperature.

   6. 1× Phosphate Buffered Saline (PBS, pH 7.4)

      Dilute 100 mL of 10× PBS pH 7.4 in 900 mL of Milli-Q water and keep at 4°C.

   7. 1× Phosphate Buffered Saline (PBS, pH 6)

      1. Dilute 100 mL of 10× PBS pH 7.4 in 800 mL of Milli-Q water.

      2. Adjust the pH to 6.0.

      3. Bring up the volume to 1 L and keep at 4°C.

   8. Ethylenediaminetetraacetic acid (EDTA, 0.5 M, pH 8)

      1. Add 146.12 g of EDTA to 850 mL of Milli-Q water.

      2. Adjust the pH to 8.0.

      3. Bring up the volume to 1 L.

      4. Filter and store at room temperature.

   9. X-Gal stock solution (25 mg/mL)

      Dissolve 150 mg of X-Gal in 5mL of DMF.

   10. Potassium hexacyanoferrate (II) trihydrate (0.5 M)

       1. Dissolve 2.11 g of Potassium hexacyanoferrate (II) in 10 mL of Milli-Q water.

       2. Store at 4°C in the dark.

   11. Potassium hexacyanoferrate (III) (0.5 M)

       1. Dissolve 1.646 g of Potassium hexacyanoferrate (III) in 10 mL of Milli-Q water.

       2. Store at 4°C in the dark.

   12. Magnesium Chloride (MgCl₂ 0.5 M)

       Dissolve 5.1 g of Magnesium Chloride Hexahydrate in 50 mL of Milli-Q water.

   13. X-Gal staining solution

       1. To prepare the SA-β-GAL staining solution (1 mg/mL X-Gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 2 mM Magnesium chloride in PBS pH 6), add 1 mL of 0.5 M Potassium hexacyanoferrate (II) trihydrate, 1 mL of 0.5 M Potassium hexacyanoferrate (III), 400 µL of 0.5 M MgCl₂ and 4 mL of 25mg/mL X-Gal to 93.6 mL of 1× PBS pH 6.0.

       2. Make a fresh solution for each experiment and keep it in the dark until use.

       Note: X-Gal concentration can be adjusted up to 1.5% to increase the staining.

   14. Ethanol 70%

       Add 300 mL of Milli-Q water to 700 mL of ethanol 100%.

   15. Ethanol 95%

       Add 50 mL of Milli-Q water to 950 mL of ethanol 100%.

   Acknowledgments

   The protocol described has been previously used and described in El Maï et al. (2020).

   We thank the Instituto Gulbenkian de Ciência histology unit for assistance with experimental planning, sample processing, and data collection, and the IGC Fish Facility for excellent animal care. IGC Fish Facility is financed by Congento LISBOA-01–0145-FEDER-022170, co-financed by FCT (Portugal) and Lisboa2020, under the PORTUGAL2020 agreement (European Regional Development Fund). MEM is a recipient of a postdoctoral fellowship from the Ville de Nice. This work was supported by the Fondation pour la Recherche Médicale FRM (EQU201903007804) and the Agence Nationale de la Recherche (ANR-21-CE14-0054) grants received by MGF.

   Ethics

   Animal experimentation: All Zebrafish work was conducted in Portugal according to National Guidelines and approved by the Ethical Committee of the Instituto Gulbenkian de Ciência and the Direção Geral de Alimentação e Veterinária (DGAV, Approval number: 010294) and in France by the Animal Care Committee of the IRCAN, the regional (CIEPAL Cote d’Azur 531 #697) and national (French Ministry of Research #27673-2020092817202619) authorities.

   Competing interests

   The authors declare that no competing interests exist.

   References

   1. Biran, A., Zada, L., Abou Karam, P., Vadai, E., Roitman, L., Ovadya, Y., Porat, Z. and Krizhanovsky, V. (2017). Quantitative identification of senescent cells in aging and disease. Aging Cell 16(4): 661-671.
   2. Collins, C. J. and Sedivy, J. M. (2003). Involvement of the INK4a/Arf gene locus in senescence. Aging Cell 2(3): 145-150.
   3. Coppé, J. P., Patil, C. K., Rodier, F., Sun, Y., Munoz, D. P., Goldstein, J., Nelson, P. S., Desprez, P. Y. and Campisi, J. (2008). Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol 6(12): 2853-2868.
   4. Dimri, G. P., Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C., Medrano, E. E., Linskens, M., Rubelj, I., Pereira-Smith, O., et al. (1995). A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A 92(20): 9363-9367.
   5. El Maï, M., Marzullo, M., de Castro, I. P. and Ferreira, M. G. (2020). Opposing p53 and mTOR/AKT promote an in vivo switch from apoptosis to senescence upon telomere shortening in zebrafish. Elife 9: e54935.
   6. Gorgoulis, V., Adams, P. D., Alimonti, A., Bennett, D. C., Bischof, O., Bishop, C., Campisi, J., Collado, M., Evangelou, K., Ferbeyre, G. et al., (2019). Cellular Senescence: Defining a Path Forward. Cell 179(4): 813-827.
   7. Henriques, C. M., Carneiro, M. C., Tenente, I. M., Jacinto, A. and Ferreira, M. G. (2013). Telomerase is required for zebrafish lifespan. PLoS Genet 9(1): e1003214.
   8. Lee, B. Y., Han, J. A., Im, J. S., Morrone, A., Johung, K., Goodwin, E. C., Kleijer, W. J., DiMaio, D. and Hwang, E. S. (2006). Senescence-associated beta-galactosidase is lysosomal beta-galactosidase. Aging Cell 5(2): 187-195.
   9. Li, T., Liu, X., Jiang, L., Manfredi, J., Zha, S. and Gu, W. (2016). Loss of p53-mediated cell-cycle arrest, senescence and apoptosis promotes genomic instability and premature aging. Oncotarget 7(11): 11838-11849.