for haemolymph

1. Collect the haemolymph into a calibrated microcapillary as described for the JH radioimmunoassay (see section 3.1.1).

2. Note its exact volume.

3. Transfer the haemolymph into an Eppendorf tube containing cold methanol in a 1:100 volume ratio (100 µl methanol per µl haemolymph).

4. Keep at 4oC for a couple of hours (best overnight).

5. Samples can be stored at -20oC in these Eppendorf tubes, as ecdysteroids will not adhere to plastic the way JH does.

6. Centrifuge the tubes to pelletize any remaining impurities.

Most haemolymph samples can be assayed directly by RIA. If there are problems, try purifying free ecdysteroids by chromatographic separation (see section, below).