Whole Mounts of Pupal Retinas
- Age pupae the appropriate length of time. Time zero should be at the white prepupal stage. Transfer pupa to dissecting dish and with number 11 scalpel, cut away the anterior and posterior ends of the pupal case, being careful not to cut the pupa itself.
- With forceps, grab the pupa by its hindend and pull it out of the pupal case, like pulling an arm out of a sleeve. Transfer the pupa to a puddle of cold 0.1M Phosphate pH 7, and make a small sagittal incision through the head.
- Place the tip of a P20 eppendorf pipet (set at 3 ml) right next to the incision and pipet back and forth. Alot of mush will be sucked out but also the retinas attached to the brain hemispheres will come out. Often the hemispheres become separated from each other, leaving two structures that resemble toadstools. The flat "top" of the toadstool is the retina. With the P20, pipet the complex into PLP fix in a petri dish, and fix for 40 minutes.
- All manipulations must be done with the P20 pipet. It is very tricky to handle the brain-retina complex with forceps or a hook. Transfer tissue to PS buffer (0.1M Phosphate pH 7, 0.1% saponin) and wash twice for 10 minutes each. Transfer to solution containing primary antibody diluted into PS + 5% goat serum and incubate 1 hour. All steps including this one can be done in a microtiter well.
- All subsequent steps in 5. should be done in the same microtiter well by replacing the old solution with new by P20. The tissue is very sticky at this point and it's best not to touch it too much. Wash for 5 X 5 minutes in PS and incubate 1 hour in secondary antibody diluted in PS + 5% goat serum. Wash for 5 X 5 minutes in PS and carry out DAB reaction as with eye discs. Wash thoroughly in PS.
- Transfer by P20 to eppendorf tube and if necessary, postfix 5 minutes in 2% OsO4. Dehydrate through an ethanol series and clear in methyl salicylate. Dissect brain away from retina and mount retina in methyl salicylate.
original text by Richard Carthew