Fluorescent Immunostaining

1. Fly growth medium: Use bottles with rich medium (e.g., [1] 80 g fresh yeast, 80 g wheat flower, 11 g agar, 50 mL Moldex, and water to 1 L final volume, or [2] 8 g agar, 18 g dried yeast, 10 g soybean meal, 7 g molasses, 80 g malt extract, 80 g cornmeal, 6.3 mL propionic acid, and water to 1 L final volume).

2. Dissection stainless-steel tweezers no. 5.

3. Tris-HCl, pH 7.5, Tris-HCl, pH 8.5, Triton X-100 and 1 N KOH, Nonidet P40 (NP-40), Tween-20, glycerol, NaCl powder, nonfat dry milk powder (can be obtained directly from grocery stores).

4. Poly-L-lysine-coated slides. They can be purchased commercially (e.g., Merck Eurolab, cat. no. 711909) or prepared in the laboratory. For laboratory preparation, start with 100-200 slides in racks. Soak the slides in corrosive detergent solution for 2 h. Wash in running tap water for 2 h. Wash twice in bidistilled water (ddH2O). Dip twice in 95% ethanol. Air-dry. Dip the slides in poly-L-lysine solution (slide adhesive solution, 0.1% [w/v] in water; Sigma-Aldrich, P 8920). Withdraw the rack; the solution should cover the slides uniformly and stay on the slides. Air-dry the slides.

5. Standard cover slips, 22 x 22 mm (Corning or others).

6. Latex gloves.

8. Solution 1: 0.1% Triton X-100 in phosphate-buffered saline (PBS), pH 7.5. Dissolve in PBS from a 10% Triton X-100 (w/v in ddH2O) stock solution. Store at room temperature (RT) in the dark.

9. 37% Formaldehyde stock solution: Weigh 1.85 g of paraformaldehyde and dissolve in a final volume of 4.93 mL ddH2O. Add 70 ||L of 1 N KOH and dissolve by heating to 60°C. Store in 100-|L aliquots at -80°C. This solution is stable for several months. To test its stability, thaw aliquots by boiling for 1 min. If a precipitate forms or the color of the solution becomes brownish, it should be discarded and prepared again.

10. Solution 2: 3.7% Formaldehyde, 1% Triton X-100 in PBS, pH 7.5. Prepare by adding 400 |L of PBS (pH 7.5) and 50 |L of 10% Triton X-100 to 50 |L of 37% formaldehyde stock. This solution should be made fresh every 2-3 h.

11. Solution 3: 3.7% Formaldehyde, 50% acetic acid. Prepare by adding 50 |L of 37% formaldehyde stock solution to 200 |L of ddH2O and 250 |L of glacial acetic acid. Prepare fresh every 2-3 h.

12. Slide racks.

13. Liquid nitrogen.

14. Diamond-tip pen.

15. Razor blade.

16. Protective glasses.

17. Methanol.

19. Block solution: 3% Bovine serum albumin (BSA) (fraction V; Sigma-Aldrich, A-2934), 0.2% (w/v) NP-40, 0.2% (w/v) Tween-20, and 10% w/v nonfat dry milk (from any grocery store) in PBS. To make 500 mL: Add 15 g BSA powder to 440 mL of PBS and dissolve by magnetic stirring. Add 1.0 g NP-40 and dissolve by stirring. Add 1.0 g of Tween-20 and dissolve by stirring. Add 50 g of nonfat dry milk and dissolve by stirring. The solution should have a homogeneous milky aspect. Store at -20°C in 50-mL aliquots.

20. Wash solution A: 300 mM NaCl, 0.2% (w/v) Tween-20, and 0.2% (w/v) NP-40 in PBS. To make 1 L, dissolve the following in 1 L of PBS: 9.55 g NaCl, 2.0 g NP-40 and 2.0 g Tween-20.

21. Wash solution B: 400 mM NaCl, 0.2% (w/v) Tween-20, 0.2% (w/v) NP-40 in PBS. To make 1 L, dissolve the following in 1 L of PBS: 15.4 g NaCl, 2.0 g NP-40, and 2.0 g Tween-20.

22. 200X 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, D 9542) stock solution: For a 200X stock, dissolve DAPI in 180 mM Tris-HCl (pH 7.5) at 0.1 mg/mL. Store frozen in 100-|L aliquots and protect from light. Working solution can be made by diluting an aliquot of stock in PBS or in 50 mM Tris-HCl, pH 7.5, at a concentration of 0.5 |g/mL.

23. Mowiol mounting medium: Add 2.4 g Mowiol 4-88 (Calbiochem no. 475904) to 6 g of glycerol and 6 mL of H2O. Mix for 3 h and add 12 mL of 0.2 M Tris-HCl, pH 8.5, and incubate for 30 min at 60°C with mixing. Pellet insoluble material by centrifugation at 5000g for 15 min. Add Diazabicyclo[2,2,2]octane (DABCO) (Merck no. 803456) to 2.5% (w/v) as an antibleaching agent. Make 500-|L aliquots and store at -20°C.

2.2. Postfixation by Formaldehyde or EGS

1. 3.7% Formaldehyde in PBS: Prepare fresh from 37% stock solution (see Subheading 2.1., item 9); add 50 |L of stock to 450 |L of PBS.

2. 50 mM Ethylene glycol-bis[succinic acid N-hydroxysuccinimide ester] (EGS) (Sigma-Aldrich, E-3257). Prepare fresh in PBS.

2.3. Fluorescent In Situ Hybridization

1. Hybridization solution: 2X SSC, 10% (w/v) dextran sulfate, 50% formamide, 0.8 mg/mL salmon sperm DNA. 20X SSC stock solution is 3 M NaCl, 0.3 M

sodium citrate, pH 7.0; 50% dextran sulfate stock solution is prepared by stirring 25 g of dextran sulfate in 50 mL final volume of sterile H2O (store at 4°C). Salmon sperm DNA solution at 8 mg/mL is prepared according to ref. 17. To make 10 mL of hybridization solution, mix 2 mL of 50% dextran sulfate, 5 mL formamide, 1 mL of DNA solution, and 1 mL of 20X SSC. Add water to 10 mL. Store in 500-|L aliquots at -20°C.

2. Detection solution: 50 mM Tris-HCl, pH 7.5, 4% (w/v) BSA. Prepare before use by dissolving 80 mg of BSA powder in 2 mL of 50 mM Tris-HCl.

3. BioNick labeling system (Gibco-BRL, cat. no. 18247-015).

7. Fluorescein isothiocyante (FITC)- or Texas Red-conjugated streptavidin (Vector Laboratories, cat. nos. SA-5001 and SA-5006).

8. Biotinylated antistreptavidin antibodies (Vector Laboratories, cat. no. BA-0500; optional).

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