>I'm a grad student starting project involoving confocal study of
>neuropeptides in the brain of the insect Rhodnius prolixus.


Dear Kathy T,

We have successfully used the following protocol to map neuropeptides in
Drosophila and Lucilia nervous systems.  Contact us directly for further
advice.
Cheers,
Eric Hines
Microscopy Centre
CSIRO Entomology
Canberra.


HINES, E.R., BROOKS, E.M., SUTHERLAND, T., and EAST, P.D. (1992).
Immunocytochemical Characterization by Confocal Laser Scanning Microscopy
of FMRFamide-like Neuropeptides in Lucilia and Drosophila. J.
Computer-Assisted Micros (Sept 1992).
DUVE, H., THORPE, A., SCOTT, A., JOHNSEN, H., REHFELD, J. F., HINES. E. and
EAST,P.(1995).  The sulfakinins of the blowfly Calliphora vomitoria.
Peptide isolation, gene cloning and expression studies.  Eur. J. Biochem.
232:633-640.


Ab Staining of Drosophila and Lucilia Tissue

Hines/East after Patel


# Dissect under PBS in large plastic petri dish pre-rinsed with PBT to
prevent adhesion.(Transfer to fix with forceps)

# Fix in 4% paraformaldehyde in PBS or PO4 with 170mM NaCl at r.t./15
mins.(In plastic petri dish pre-rinsed with PBT. Transfer to screw-top
scintillation vials with pre-rinsed pipette. Allow brains or embryos to
settle for 5 mins before pipetting)

#Wash 2x 5 mins in PBS to wash out paraformaldehyde (keep vials upright,
spin by hand to mix)

#Permeablize by either

Detergent Treatment
60 mins PBT

or

 Methanol Treatment
5 mins 70% MeOH in PBS
60 mins 100% MeOH in PBS
5 mins 70% MeOH in PBS
2x5 mins PBS


#Wash 2x 5 mins in PBT (vials upright - hand spin)

#Incubate 30 mins in 100ml of PBT + N (vials upright on shaker)

#Add 100ml 1o Ab supernatant. Incubate 2 hrs at 20oC plus o'night at 4oC,
i.e. a 1 : 1 dilution of 1o Ab. (Use manufacturers recommended final
dilution. Vials upright. Incubation time can be extended up to 3 days of
12hrs r.t./12hrs 4oC)

#Wash 3x 5mins in PBT  (vials upright, hand spin)

#Wash 2x 45mins in PBT (vials upright on shaker)

#Incubate 30mins in 100ml of PBT + N (vials upright, hand spin)

#Add 100ml 2o Ab goat anti rabbit/mouse FITC conjugated. Dilution should be
in PBT + N (remember we have 1:1 dilution plus Ab dilution)

#Incubate at room temp for 2hrs plus overnight at 4oC

#Wash 3x 5mins in PBT (vials upright, hand spin)

#Wash 4x 30mins in PBT (caps wetted, vials filled, complete inversion)

#Rinse 3x 5 mins in PBS

#Clear tissue in glycerol or methyl salicylate after dehydration. (place
embryo/brain into 50% glycerol for 2hrs then 70% glycerol overnight )

#Mount in anti-fade/glycerol



Stocks:

#PBT is 1x PBS  with.0.2% BSA
0.1% Triton X-100

#Normal Goat Serum
Use PBT + N
4mls PBT + 200ml NGS (NGS must be previously heat treated at 56oC for
30mins and aliquots frozen at -20oC)
Spin before dilution if not purified NGS
Sterile filter after dilution if not purified.

#Anti-fade Mounting Media for FITC
Dissolve 0.1 - 0.01% p-phenylenediamine (Sigma #P1519)
in 50% glycerol in PBS.(We are presently using 0.01% but this varies
between batches.
Aliquot and store frozen -80oC. If it goes dark throw it out)

#PBS is made up as 10x stock and diluted for use.
For 2.5 litres of 10x stock
28.75 g Na2HPO4
200 g NaCl
5 g KCl
5 g KH2PO4

#Formaldehyde is made up fresh daily from para-formaldehyde (pF)
Dissolve pF in small amount of dH2O. (Heat to 60oC and add NaOH)
Make up to volume with PBS and pH to 6.8 to 7.2