Materials and Reagents

1. Aluminum foil
2. Eppendorf tubes
3. Pipettes range of 0-10 μl, 10-200 μl, 200-1,000 μl
4. Microscope mounting Slides (Sigma-Aldrich, catalog number S8902) 
5. Cover slips (Thermo Fisher, catalog number 102455)
6. Albumin Bovine, Fraction V (MP biomedicals, catalog number: 0216006980)
7. CpG Methyltransferase (M. SssI) (Supplied with Buffer and S-adenosylmethionine [SAM])- (New England Bio-labs (NEB), catalog number: M0226L)
8. Alexa Fluor 568 goat anti-mouse IgG (H + L) (Invitrogen, catalog number: A11031)
9. 5-Methylcytosine (5-mC) antibody (mAb) - clone 33D3 (Active Motif, catalog number: 39649)
10. Schneider's Drosophila Medium (Biological Industry, catalog number: 01-150-1A)
11. Dulbecco's Phosphate Buffered Saline (DPBS) (10x) (Biological industries, catalog number: 02-023-5A)
12. Pierce^TM 16% Formaldehyde (w/v), Methanol-free (Thermo Scientific^TM, catalog number: 28906)
13. Heptane (Sigma-Aldrich, catalog number: 246654)
14. Triton X-100 (Sigma-Aldrich, catalog number: X100)
15. 32% Hydrochloric acid (Bio-Lab, catalog number: 084605)
16. Borax anhydrous (Sigma-Aldrich, catalog number: 71997)
17. DAPI-for nucleic acid staining (Sigma-Aldrich, catalog number: D9542-1MG)
18. Fluoromount-G (Southern Biotech, catalog number: 0100-01)
19. HCl
20. Fix-I solution (1 ml) (see Recipes)
21. PBX (see Recipes)
22. PAT (see Recipes)
23. Pre-reaction Buffer (1 ml) (see Recipes)
24. M. SssI Reaction Buffer-100 μl (25 U) (see Recipes)
25. Fix-II solution (1 ml) (see Recipes)
26. PBT (see Recipes)
    

Equipment

1. Incubator for rearing Drosophila at consistent temperature of 29 °C
2. Stereomicroscope with light source for dissection (Leica Microsystems, model: MZ75)
3. Dumont tweezers #5 Biology 0.05 x 0.02 mm Inox (magnetic, FST11252-20)
4. Watch Glasses square for dissection (Carolina, catalog number: 742300) 
5. Analog Orbital Shaker (ELMI S-3.02 10 L) 
6. Confocal microscope (Zeiss, model: LSM-700)
   

Procedure

1. Flies collection
   Drosophila flies are reared at 22 °C on standard medium. To prevent gender differences, collect only female flies at relevant age. For collecting young adults, we used 3-10 days old flies that were transferred to 29 °C for two days for activating the GAL4/Gal80 system and UAS-RNAi for gene knockdown. For more details regarding the GAL4/GAL80 system, see Salmeron et al. (1990) and Brand and Perrimon (1993). 


2. 5mC methylation assay
   1. Dissect guts in watch glasses squares filled with cold Schneider medium. Transfer dissected guts to an Eppendorf tube containing 0.5 ml Schneider medium. Eppendorf tubes with dissected guts are kept on ice. Collect 8-10 guts per Eppendorf tube. For detailed gut dissection, see an excellent midgut protocol (Nawrot-Esposito et al., 2017). 
   2. Remove Schneider media and add 1 ml cold DPBS to wash dissected guts (replace media only without incubation time).
   3. Fix: Replace DPBS with 1 ml of freshly made "Fix-I solution" (Recipe 1), this will form two phases of liquids: the upper phase is Heptane and lower phase is the formaldehyde/DPBS solution at the bottom (See Figure 3 “Fix”). Rotate for 20 min on an orbital shaker at RT.
      Note: Over-crosslinking can cause reduced diffusion and accessibility of the enzyme to the nuclei. 
      
      
      Figure 3. Typical Eppendorf tube after formation of the two phases
      
      
   4. Gently remove all the liquid solutions (the 2 phases) leaving only the dissected tissue at the bottom. Be sure to remove all heptane, also from the cap.
   5. Wash guts three times with 1 ml DPBS, in each wash rotate the guts for 15 min at RT. 
   6. Permeabilization: Permeabilize the tissue by incubating with 500 µl of 0.5% Triton X-100 in DPBS for 1h on an orbital shaker in Eppendorf tubes, at RT (we recommend evaluating different permeabilization conditions [e.g., 1 h, 2 h and triton concentrations (0.5%, 0.6%, 0.7%)]).
   7. Gently remove Triton solution, and wash for three times with DPBS, in each wash rotate the guts for 15 min at RT.
   8. Block in 1 ml "PAT solution" (Recipe 2) overnight at 4 °C on an orbital shaker.
   9. Wash twice with freshly prepared 250 μl of "Pre-reaction Buffer "(Recipe 3), 15 min each wash. 
   10. Methylation reaction: Gently remove "Pre-Reaction Buffer” and resuspend in 50 µl of M. SssI reaction buffer (Recipe 4) and incubate for 1 h at 25 °C, on an orbital shaker.
       Important: Titer the reaction conditions time (e.g., 1 h, 5 h, 24 h) and enzyme amount (25 U, 50 U) for optimal results.
   11. Termination: Add 1 ml of 2 N HCl and rotate on an orbital shaker at room temperature for 30 min. This step stops the M. SssI reaction and denatures the DNA.
   12. Remove HCl solution and neutralize with 0.5 ml of freshly prepared 100 mM Borax (dissolved in water) and rotate on an orbital shaker for 5 min at RT.
   13. Remove Borax and wash twice with 1 ml DPBS, rotate the guts for 15 min at RT each wash.
   14. Fix with freshly prepared "Fix-II solution" (Recipe 5) and rotate for 10 min. 
   15. Remove Fix-II and wash twice with 1 ml "PBX solution" (Recipe 6), rotate the guts for 15 min at RT each wash.
       Note: Evaluate different time points (10, 15, 20 min) as over-crosslinking could affect antibody penetration.
   16. Remove PBX and block with 1 ml "PAT solution" (Recipe 2), for 30 min rotating at RT. 
   17. Remove blocking solution and add diluted anti-5mC primary antibody in "PAT solution" (Recipe 2), to a concentration of 1 μg/ml (1:1,000). Incubate overnight at 4 °C on an Orbital shaker. 
   18. Remove primary antibody and wash three times with "PBT solution" (Recipe 7), rotating for 15 min each wash at RT.
   19. Remove PBT and incubate in diluted Secondary Fluorescent anti-mouse (1:1,000) together with DAPI (1:1,000) (for DNA staining) in "PAT solution" (Recipe 2), cover the samples with aluminum foil or in a light protected box, and rotate on orbital shaker for 1 h in the dark. 
   20. Remove Secondary antibody and DAPI solution and wash three times with "PBT solution" (recipe 7), 15 min each wash while rotating in light protected environment (box or aluminum foil) at RT.
   21. Wash twice with 1 ml DPBS, rotate the guts for 15 min at RT each wash.
   22. Mount in a Fluoromount-G (25 ml) on slide and keep at 4 °C until confocal microscopic analysis.
   23. Visualize under a confocal microscope, signal intensity in cells can be quantified. For quantification we suggest counting several hundreds of cells derived from different biological repeats, and in each scoring 5mC positive polyploid cells. 

Recipes

Prepare each solution fresh and keep no longer than a week at 4 °C or on Ice.

1. Fix-I solution (1 ml)
   75 μl 16% EM-grade formaldehyde
   12.5 μl 10x DPBS
   112.5 μl distilled water
   600 μl Heptane
2. PAT
   1% BSA in PBX
3. Pre-reaction Buffer (1 ml)
   899.5 μl distilled water
   100 μl 10x NEB buffer (New England Biolab MsslI buffer)
   0.5 μl SAM (S-adenosyl methionine from NEB, supplied with the enzyme)
4. M. SssI Reaction Buffer-100 μl (25 U)
   6.25 μl M. SssI
   83.25 μl distilled water
   10 μl 10x NEB
   0.5 μl SAM
5. Fix-II solution (1 ml)
   50 μl 16% EM-grade formaldehyde
   150 μl 1x DPBS 
6. PBX
   0.1% Triton X-100 in 1x DPBS
7. PBT
   0.1% BSA in PBX
   

Acknowledgments

This original protocol was used in Rincon-Arano et al. (2012). The research was supported by Israel Science Foundation (ISF) (Grant 739/15), and the Rappaport Institute for Research in the Medical Sciences and Flinkman-Marandi cancer research grant to AO.

Competing interests

We declare that there is no financial or non-financial conflict of interest, or competing interests related to this work to any of the authors.

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