Drosophila Testis FISH

RNA FISH on Drosophila testes

General points:

I do all the liquid handling steps in baskets held in 48-well plates, so my first step would be “transfer tissue to baskets in a 48-well plate”. I find this is easiest to do by pipetting all your fixed testes onto a shallow dish – I like petri dish lids – and then pipetting them into the baskets. You can do the whole process tubes, though – it’s just a pain. 

Technically, you can combine probes (and their respective antibodies) with any tyramide. However, if you are using both Fluorescein tyramide, you cannot hybridize a FITC-conjugated probe *after* you’ve done that development – the antibody will stick there. The order I typically like is:

D1: Hybridize DIG and FITC probes

D2: Wash into anti-FITC

D3: Develop with Rhodamine and wash into anti-DIG

D4: Develop with Fluorescein, mount. 

But you could also develop the anti-DIG with Cy5 if you are, for example, combining it with a antibody in GFP. Or, if you’re only using one probe, the strongest single combination is usually anti-DIG with Rhodamine. Some optimization is sometimes required on a per-probe basis. 

Day 1:

Solutions to make for the first day. 

Proteinase K Solution (volumes for 10mL, adjust as necessary; make immediately before relevant step)

       4 μg/mL Proteinase K (2 μL of a 20 mg/mL stock)

       0.1% SDS (100 μL of a 10% stock): 

       In PBST (9.9 mL)

Phybe Solution (volumes for 30mL; can be made in advance and stored at 4°): 

       50% deionized Formamide (15 mL of 100% stock)

       5x SSC (15mL of 20x stock)

       1mg/mL yeast RNA (600 μL of 50mg/mL stock, dissolved in formamide)

       1% Tween-20 (3 mL of 1% stock)

       In ddH₂O (3.9 mL)

Hybe solution (volumes for 6 mL; can be made in advance and stored at 4°):

       50% deionized Formamide (3 mL of 100% stock)

       5x SSC (1.5 mL of 20x stock)

       1 mg/mL yeast RNA (120 μL of 50mg/mL stock)

       1% Tween-20 (600μL 10% stock)

       5% Dextran Sulfate (600 μL of 50% stock)

       In ddH₂O (180 μL)

Liquid steps. All at RT, shaking, unless otherwise noted.

1. 50/50 MeOH/PBST, 5’
2. PBST, 3X, 5’ each
3. ProtK solution, 6’ (absolute max; begins with 1^st basket transfer; fill next wells in advance)
4. 4% Formaldehyde, 10’(maximum; fixes tissue and removed ProtK; FA waste)
5. PBST, 3X, 5’ each
6. 50/50 PBST/Phybe, 10’
7. Phybe, 1-2 hours, 56° (Shake N’ Bake, verify temp). Cover with sticky foil (press with kimwipe to avoid papercut) + lid to prevent evaporation

Dilute and denature your probes:

1. Dilute probes 1:800 in Hybe – one dilution per sample. (Ex. #1: aub-DIG + fzo-FITC; # 2: tj-DIG + vas-FITC)
2. Incubate Hybe dilutions at 72° for 5’ (to disrupt H bonds à linearize from RNA secondary structures)

Hybe with probes:

1. overnight (at least 16 hours) @ 56°

Day 2:

Solutions to make

Block (can be made in advance, filtered & stored at 4° for up to 2 weeks)

          10% Roche Western Blocking Reagent in PBST (shake stock before pipetting bc will settle)

Steps

1. Phybe, 10’, 56° (FA waste)
2. Phybe, 20’, 56° (FA waste)
3. 50/50 2x SSC/Phybe, 10’, 56° (FA waste)
4. 50/50 2x SSC/Phybe, 20’, 56° (FA waste)
5. 2x SSC, 10’, 56°
6. 2x SSC, 20’, 56°
7. 0.2x SSC, 10’, 56°
8. 0.2x SSC, 20’, 56°
9. Take the plate out of the incubator/oven and let it cool to room temperature on nutator
10. PBST, 2X, 10’ each
11. Block, 30’(RT)
12. Dilute first antibody in block. Both anti-DIG and anti-FITC can be used at 1:1500
    1. If 2 mL total, then 1.33 μL antibody
    2. Seal with sticky foil
13. Antibody dilution, overnight, 4°

Day 3:

Solutions to make      

Sodium azide should be freshly diluted in PBST before use (ONLY IF PERFORMING double or triple FISH)

TSA Buffer (volumes for 1 L, adjust as necessary; can be made in advance & stored at RT for years)

       100 mM borate (6.185 g Boric Acid)

       2M NaCl (116.88 g NaCl – NOT A TYPO, this is a salty solution).

       In ddH­­­₂O (fill beaker to just under 1 L and top up to 1 L after adding all NaOH pellets)

       pH to 8.5 with NaOH 

4-IPBA (iodophenylboronic acid) solution (can be made in advance and stored at -20° forever)

       20mg/mL of 4-IPBA in DMF (recommend to make 1-2 mL at a time, store at -20°)

Tyramide solution (volumes for 2 mL, adjust as necessary; must be made fresh immediately before step & kept in dark)

       0.003% H₂O­₂ (1 μL of 30% stock in 100 μL TSA then 20 μL of this dilution)

       1:1000 of 4-IPBA solution (2 μL)

       ~1:1000 tyramide solution (should be empirically determined for each tyramide stock. 

       The Cy5 tyramide often needs to be used a somewhat higher concentration – around 1:500). (2 μL)

       In TSA Buffer (2 mL)

NaN₃ solution (volumes for 2 mL, adjust as necessary; only for 2x/3x FISH; must be made fresh; NaN₃ waste)

       1% Sodium Azide (400 μL of 5% stock)

       In PBST (1600 μL)

Steps:

1. PBST, 7X, 5-20’ each (increase time by 2-3 minutes per wash)
2. TSA buffer, 10’ (okay to exceed)
3. Tyramide solution, 10’ (cover tube in foil prior to making solution)
   1. Samples will be light sensitive from this point on, leave in dark
4. PBST, 3X, 5’ each

    5a.  If only doing one probe, mount here. 

    5b.  If doing one probe and then performing IF:

             a. PBST, 3x, 5’ each (for a total of 6 PBST washes)

             b. 0.3% BSA, 1 hr

             c. Primary Antibody diluted in 0.3% BSA at appropriate concentration, overnight 4°.

    5c.  If staining with two probes, continue:

             d. Sodium Azide solution, 90’

             e. PBST, 7X, 5’ each

             f.  Block, 30’ 

             g. Dilute second antibody in block. Both anti-DIG and anti-FITC can be used at 1:1500

             h. Antibody dilution, overnight, 4° (cover with sticky foil, lid + box)

Day 4 (if staining with two probes)

1. PBST, 7X, 10’ each
2. TSA buffer, 10’ (okay to exceed)
3. Tyramide solution, 10’
4. PBST, 3X, 5’ each
5. DAPI stain and mount.
   1. Fill the lid of a little petri dish with PBS
   2. Transfer one basket to the PBS and stabilize it with forceps around the diameter
   3. Pipette up and down to transfer tissue from the basket to a slide with a P200
   4. Move the empty basket to a separate container and set aside to be cleaned later
   5. Remove excess PBS from the slide and add 1-2 drops of Vectashield with DAPI
   6. Add coverslip and seal with nail polish
   7. Repeat for each basket
6. If doing IF:
   1. PBST, 7x, 5-20’ each (increase time each wash)
   2. 0.3% BSA, 1 hr
   3. Secondary Antibody diluted in 0.3% BSA at appropriate concentration, overnight 4°.

Day 5 (if staining with two probes + IF)

1. PBST, 7x, 5-20’ each (increase time each wash)
2. Mount in Vectashield with DAPI. Let sit on slides for a bit before imaging (overnight is best).