2) Wait the appropriate amount of time before dissecting the pupae. Pupae that are less than 40 hrs. old are more mushy and therefore more difficult, but not impossible, to dissect than those that are older. There is an excellent discussion of the various stages of pupal retina development described in Cagan and Ready (Dev. Bio. 136, pgs. 346-362).
3) Remove the hard exterior cuticle by cutting the posterior 1/6-1/8th of cuticle off. This can be done with a surgical blade by carefully cutting through only the hard exterior cuticle all the way around the pupae somewhat like using a can opener.
4) Inside the hard exterior is a varying degree of mush encased in a soft transparent cuticle layer. After step 3 the posterior end of the pupae will be visible through the hole made in the exterior cuticle. Grab both the posterior end of the pupae and the anterior end of the hard external cuticle with #5 forceps gently pulling the pupae from inside the hard external cuticle. Some mush will probably leak out from the posterior of the pupae during this step but the anterior end will remain intact.
5) Place the liberated pupae immediately into PBS or 0.1 M phosphate pH 7.2 for further dissection. Using a surgical blade cut a hole, from dorsal to ventral, in the anterior edge of only the transparent soft cuticle. Be careful not to smash the anterior end too much. Once a small hole is created in the cuticle it can be enlarged by inserting the tip of the surgical blade in the hole and cutting in the dorsal and ventral directions.
6) With #5 forceps, give one or more forceful squeezes to the pupae starting near the middle and working toward the anterior. After the first couple of squeezes, the brain with attached retina and alot of mush should pop out the hole created in the anterior of the pupal cuticle. Wash the mush away with a yellow pipet tip attached to a P20. The brain and retina will not be obvious until the mush has been washed away. At 40 hrs. of pupal development the brain and retina look sort of like two mushroom tops attached by their stalks.
7) In order to transfer the brain retina complex from one vessel to another, spear the
complex in the middle of the brain with a very fine tungsten needle.
Antibody Staining of Pupal Retina/Brain Complexes:
1) The pupal retina;/brain complex is dissected as above in 0.1 M PO4 or PBS and
then transfered into an eppendorf tube containing 700 µl of fixation solution.
Transfer the complex by spearing the middle of the brain with a fine tungsten
needle. Some people prefer to transfer the complex by pipetting it into a P20 tip.
Two fixation solutions which work well on fly retinas are (a) PLP (see eye disc
staining protocol) and (b) PEMP (0.1 M Pipes pH 7.0, 2.0 mM EGTA, 1.0 mM
MgSO4, 4% paraformaldehyde). Fix the retina/brain complex for 30-40 min. on ice.
2) Remove the fixation solution by aspiration and add 1 ml of ice cold PBT (1 x PBS, 0.2% BSA, and 0.1% Triton) or PSB (0.1 M PO4, 0.1% saponin, 0.2% BSA). The PBT buffer will provide better penetration of antibodies as triton is a stronger detergent than saponin. However, some membrane antigens are soluablized by triton so be careful in your choice of wash solution.
3) Wash the retina/brain complexes at least 30-40 min. with a minimum of 5 more 1 ml changes of wash solution (PBT).
4) Remove the last wash and replace it with 100-200µl of your primary antibody dilution in PBT + 5-10% serum. The appropriate primary antibody dilution required for your antibody must be determined empirically. Incubate retina/brain complexes in primary antibody on ice in the eppendorf tube for at least 2 hrs.
5) Remove the primary antibody and perform 3 x 10 min. washes with 1 ml of ice cold PBT solution.
6) Replace the last wash with 100-200 µl of diluted secondary antibody in PBT + 5-10% serum. Our lab uses a Goat anti-mouse HRP conjugated second antibody obtained from New England Bio Labs. We use this antibody diluted 1/250-1/500. Jackson Immuno Research sells comparable reagents conjugated with biotin, HRP, Alkaline Phospatase, and fluorescent chromophores. The choice of secondary antibodies should be based on the sensitivity required and the application. Incubate complexes in secondary antibody for 1-2 hrs.
7) Wash the complexes 3 x 10 min. with 1 ml of ice cold PBT per wash.
cobalt intensification;
8) During the last wash prepare the HRP staining solution which should be 1 x PBS
containing 0.5mg/ml dimethylaminoazobenzene (DAB), 0.02% CoCl2.6H2O, and
0.003% H2O2. The CoCl2 and H2O2 should be added just before use of the staining
solution. Remove the final PBT wash and add the staining solution to the
retina/brain complexes. Follow the staining reaction under a microscope and stop
it by adding two 1 ml washes of cold PBS. Unused DAB solution should be
disposed of in bleach.
9) Dehydrate the stained complexes by passing them through an ethanol series (30%, 50%, 70%, 90%, 2 x 100%) and then mount the retinas in DPX (Fluka Chemicals). Once placed in DPX the retinas can be dissected from the brain hemispheres and mounted flat.
cobalt sulfide;
Cobalt Sulfide Staining of Pupal Retinas:
1) Dissect the pupal retinas as described above in 0.1 M PO4. The dissection and all
the other fixing and staining steps are carried out in puddles on a Sylgard
dissecting dish. Transfer the retina/brain complexes from one puddle to the next
by spearing the brain with a tungsten needle.
2) Fix the retina/brain complexes for 5-15 min. at r.t. in 2% gluteraldehyde, 0.1 M PO4 (pH 7.2).
3) Dip the speared retina in a puddle of 0.1 M PO4 briefly and then soak the retinas in 2-4% Co(NO3)2.6H2O for 5 min. at r.t.
4) Dip the speared retina/brain complex into water for a brief wash.
5) Transfer the retinas into 1-2% (NH4)2S and incubate at r.t. until the retinas turn black. If the retinas do not turn black use the higher concentrations of the cobalt and (NH4)2S reagents.
6) Wash the retinas briefly by dipping the speared complexes into water.
7) Mount the retinas in 80% glycerol or Aqua-Poly/Mount (Polysciences). Dissect the retina from the brain and mount it flat.
8) See Cagan & Ready (Dev. Bio. 136, pgs. 346-362) for an excellent description of pupal retina development.
Sylgard 184; Silicone Elastomer Kit (Dow Corning) is available from
K.R. Anderson,
2800 Bowers Ave.
Santa Clara, CA 95051
408-727-2800.
You can add India ink for a dark work surface.