Staining larvae with POP-1 monoclonal antiserum

Modified from Rueyling Lin 1/99

1. Prepare poly-lysine coated slides (ala Bowerman)

Poly-L-lysine (Sigma, MW 70,000-150,000  P-1274)  Add ~1 mg chunks of ply-lysine to 1.5 ml microfuge tube (using forceps and a scalpel or scissors to cut tiny chunks--never weighed--and add to tubes.  Wear gloves) and store at -20C.  Alternatively can use ply-L-lysine solution from Sigma (with preservatives).  On same day as staining, dissolve by vortexing in 1 ml of glass (we use double) distilled water.  Use a pasteur pipet to draw up solution by capillary action, place tip on surface of slide and allow a small drop to collect.  Spread out on slide using side of tube to spread out evenly over entire slide.  Slides should be reasonably clean--fresh out of the box or from a box opened only to make these coated slides.  Its not necessary to acid wash, although that will improve sticking (ethanol wash may be sufficient).  If slides are clean, solution should spread out evenly without beading up.  If so, place coated slide on a preheated hot plate (on high or close to high--whatever will simmer).  Keep the end of slide hanging over edge of the hot plate to facilitate picking it up.  By looking at slide from appropriate angle such that light reflects from the coating, you can see when all the water has evaporated.  Remove immediately and place in a slide rack.  Slides are good all day, but not the next day.  Always make poly-lysine solution fresh. 

2. Prepare 2% paraformaldehyde fixative

2% paraformaldehyde (.5 g in 25 ml)

60 mM PIPES
25 mM HEPES (pH 6.8)
10 mM EGTA
2   mM MgCl2

Mix up salts ahead of time.  Add paraformaldehyde to solution.  Boil gently on hot plate in the hood.  Cool to room temp before using.

3. Collect larvae--grown to desired stage from hypochlorite-treated worms

Collect larvae in 1.5 ml microfuge tube.  Spin gently and quickly to remove liquid.  Rinse once with water.

4. Add 200 l of fixative to tube with larvae.  Spin quickly and discard liquid.

5. Add appropriate amount of fixative to the tube

* Appropriate amount depends on the number of larvae.  I like to have approximately 100-200 L1 in the 10 l solution I put on slide.  When it's done right, 70% of the larvae will stick on slide.   L1 and L2 are a lot easier than older worms.

6. Pipet 10 l of worm suspension to poly-lysine coated slides and overlay worms with a coverslip (22 x 40 mm) perpendicular to the slide.

7. Wicking liquid gradually with a piece of kimwipe to flatten larvae.

** If you wick it too fast, larvae burst open and you don't get good staining.  You should see larvae turn from brownish to white/clear when you squash them right.  Once they turn whitish, they are pretty tough and don't get smashed easily.

NOTE:  I like to keep the time these larvae in fixative solution in EP tube to minimum.  Even if you have a lot of larvae, I would do them in small patches.  The larvae die but not fixed in that condition.

8. 5 min fixation in a humidifier chamber

9. Freeze the slide on a metal plate prechilled on dry ice for 5 min.  Hold down slide until frozen.  After 5 min flick of coverslip and proceed immediately to next step. 

10. Immerse slides in 100% dimethyl formamide at -20C for 5 min (First paper used abs. MeOH).

11. Immerse slides in 100% MeOH at room temperature for 5 min.

12. Rehydrate sequentially in 90%, 70%, 50%, 0% MeOH in Tris-Tween at room temp.  5 min each (?).

Tris-Tween:  100 mM Tris-HCL (pH 7.5), 200 mM NaCl, 0.1% Tween

13.  Add blocking solution ( 5% BSA in PBS) for 10 min.  Remove by wicking off liquid.

14.  Add 1/50 dilution of P4G4 (POP-1 mab) in Tris-Tween.  Incubate 12 hr (overnight) at 4C in humidified chamber. Remove

15.  Wash twice, 5 min. each in Tris-Tween at room temp.

16.  Add secondary (rhodamine-goat-anti-mouse) antibody.  Incubate 2-3 hr at room temp in humidified chamber.

17.   Wash twice, 5 min. each in Tris-Tween at room temp.  Add 20 ng/ml DAPI to the final wash.

18.  Mount in appropriate mounting media. 

A. wash 1x in 4 mM ascorbic acid in Tris-Tween (or PBS) and mount in same solution.

B. 1 mg/ml p-phenylenediamine, 10% PBS, 90% glycerol adjusted to pH 8 with 1N NaOH.  Aliquot and store in the dark at -70C. 

C. Buy SlowFade from Molecular Probes.

19.  Seal coverslip with nail polish and view

Additional solution:

PBS:

7.31 g NaCl
2.36 g Na2HPO4
1.31 g NaH2PO42H2O

Adjust pH to 7.0 and final volume to 1 liter

Can be made as 10x stock solution.