Method prepared by Grace Panganiban:

Antibody staining of larval and pupal imaginal discs

Larval or pupal heads are pulled or cut off and inverted in PBS buffer.
Inversion is best achieved by orienting the dissected head with the mouth hooks upwards (towards you) and gently pushing the mouth straight down with one forcep while anchoring the entire head with the other forcep. In this manner, the cuticle is on the inside and the discs, brain, fat, etc. are on the outside. Extraneous tissue such as digestive tract, fat, and salivary glands, are gently dissected away leaving the imaginal disc complex attached to the mouth area. It is also possible to recover the wings, halteres, and metathoraxic legs attached to the cuticle provided you are gentle in dissecting away the extraneous tissue. Dissections prior to staining as well as individual disc dissection for mounting can be done in glass depression wells or glass slides but petri dishes containing Sylguard (Dow Corning) work best.

Day 1
Fix disc complexes for 20-30 minutes in 1 ml of fixative at 4oC. Fixation time is lengthened to as long as 45 minutes if the peripodial membrane is to be dissected away. The membrane is dissected off after fixation is completed.
FIX Buffer: 0.1M Pipes pH 6.9;1 mM EGTA pH 6.9; 1.0% Triton X-100; 2mM MgSO4; 1.0% Formaldehyde (added just prior to the addition of the tissues). Note: For each antibody or combination of antibodies in double labeling experiments, it may be necessary to adjust the choice of detergent, the percentage of detergent (Triton), or the percentage of formaldehyde. The optimal conditions for anti-hairy and/or 22C10 are given.

Block tissues for 1-8 hours at 4oC.
BLOCK Buffer: 50 mM Tris pH 6.8; 150 mM NaCl; 0.5% NP40; 5nmg/ml BSA.
All subsequent steps are done in WASH Buffer.
INCUBATION/WASH Buffer: 50 mM Tris pH 6.8;150 mM NaCl; 0.5% NP40; 1 mg/ml BSA.

Antibody Labeling:
Primary antibody. Incubate tissues overnight in 200 ul of antibody solution at 4oC in individual wells (4-8 complexes/well) of a 48-well
tissue culture dish. If screens are used to hold the disc complexes,
increase volume to 300 ul. Screens are not used during the fixation or enzymatic staining steps! Antibody concentration is different for each primary antibody (e.g. anti-22C10 and anti-hairy are used@1.5 ul/200 ul wash buffer).
Day 2
Wash complexes 4 times (4 wells with 1 ml of WASH buffer in each well) over 1 1/2 hours. If screens are used, blot the screen when it is removed from the primary, secondary and tertiary antibody wells to prevent carryover.
Secondary antibody. Incubate tissues for 2 hours in 200-300 ul of secondary antibody solution at 4oC. For 22C10, goat anti-mouse rhodamine (fluorescence) or goat anti-mouse peroxidase (enzyme) is used at a concentration of 4 ul/200 ul. For anti-hairy goat anti-rabbit biotin is used at a concentration of 1 ul/200 ul.
Wash complexes 4 times (as above) over 1 hour. If fluoresceinated antibodies are used, incubation should be done in the dark. For anti-hairy, strepavidin-horseradish fluorescein (fluorescence) or
strepavidin-horseradish peroxidase (enzyme) is used at a dilution of
1ul/200ul. Wash complexes 4 times (as above) in 1 hour. These washes are done in the dark if a fluoresceinated tertiary is used. If fluoresceinated antibodies are used, after washing the discs, they
are incubated overnight in mounting media containing PDA (pphenylenediamine) to prevent quenching of the fluorescence. Thisincubation should be done in the dark. Discs are then dissected apart,mounted (in the same media) onto slides, and coverslips are sealed, after blotting excess liquid, with fingernail polish.
PDA Mount:
30 mg PDA is dissolved in 4 mls of dH20. Add 6 mls of glycerol. Mix well and freeze aliquoted at -20oC. PDA works optimally if it is made fresh weekly.
Dilute 1 ml of this mixture (fresh each time) into 5 ml 0.1M Tris-HCl pH 8.8 for working mount. KEEP STOCK AS WELL AS WORKING MOUNT IN THE DARK AS MUCH AS POSSIBLE.B.
If discs are stained with enzyme©conjugated antibodies, proceed after last wash above with two washes in 0.1M Tris-HCl pH 6.8 (5 minutes each).
For peroxidase, DAB (diaminobenzidine) is used as the substrate. A 10X DAB stock is made by adding 5 mg of DAB to 1 ml 0.1M Tris pH 6.8.
For 5 mls working (1X) DAB solution:
0.5 ml 10x DAB; 50 ul 3% CoCl2; 50 ul 3% NiCl; 3.3 ul 30% H2O2; and 4.4 mls 0.1M Tris pH 6.8 . Note: Do not add H2O2 until just prior to staining disc complexes (see below). Transfer disc complexes to empty wells and add 1 ml/well of staining solution immediately after adding H2O2. When staining of discs is sufficient, stop the reaction by adding 2 ul/well of 20% NaN3. Optimal staining is most often determined by eye at the dissection microscope for each experiment. Wash disc complexes twice in 50mM Tris-HCl pH 8.8. Incubate disc complexes overnight in mounting media (10% glycerol in 50 mM Tris pH 8.8). Discs are dissected apart (in the same media), placed on slides, and coverslips are sealed, after blotting excess liquid, with fingernail polish. If DAPI counterstain is desired, add 1:200 dilution of DAPI (stock concentration is 1 mg/ml) to the second change of the set of washes done just prior to staining with DAB. Note: DAPI quenches peroxidase 1/90 Nadean Brown and Jim Skeath c/o Carroll Lab Laboratory of Molecular Biology, University of Wisconsin-Madison,Madison, WI 53706, 608-262-7898



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