For in vitro use only!
Shipping: shipped on blue ice
Storage Conditions: store at -20 °C
avoid freeze/thaw cycles
Shelf Life: 12 months
Form: liquid (Supplied as serum)
Polyclonal antibody against the neuropeptide proctolin.
Protocol - Proctolin detection by immunocytochemistry in invertebrate nervous system
The anti-proctolin antiserum, generated against proctolin coupled to glutaraldehyde/polylysine (1:4) was tested for cross-reactivity using ELISA. No coss-reactivity was observed against 10 μg/ml of glutaraldehyde/polylysine conjugates of perisulfakinin, locustatachykinin II, FMRFamide, crustacean cardiactive peptide, adipokinetic hormone I, leucomyosuppressin, corazonin and the allatostatines, Dip-AST 2, Dip-AST 7, and Dip-AST 8.
Insects were cooled for 15 minutes and dissections were carried out in insect saline or in solution A. Ganglia or brain were exposed by opening and pinning out the dorsal cuticle, mounted dorsal- and in same cases ventral-side up on a wax coated glas disk.
Cover up the insect brain or ganglia 30 min to 120 min with one of the solutions B.
Immunocytochemistry was carried out on free-floating Vibratom sections by means of the indirect immunofluorescence immunocytochemistry. Brains or ganglia were wrapped in 5% agar and cut at 20-50 μm with a Vibratome
(optional and only for fixation with solution B1)
Vibratom sections are incubated during 10 min in solution C containing sodium borohydrite (0,1M) by stirring. Then tissue pieces are washed 5 times (15 min each) with solution C by stirring. Section are incubated during 12 hours at 4°C in solution C + 30% sucrose.
The sections are washed 3 times (15 mn each time) in solution C (for the fixation with solution B1) and in solution D (for the fixation with solution B2) at room temperture.
Application of antibody
The final dilution of the monoclonal anti-proctolin is 1:1000 in solution C or D (depending on the fixation, see above) + 0.25 % of Triton X100 + 1% goat serum + 3% milk powder (without fat) + 0,25 % BSA.
A dozen of sections can be incubated with 2ml of antibody solution overnight or 48 h at 4°C, by stirring. Then the sections are washed 3 times 30 minutes each with solution D for both fixations by stirring.
Sections are incubated with 1:600 dilution of Carbocyanin 3(Cy-3)-goat anti-mouse complex (Jackson ImmunoResearch Laboratories, Inc.) in solution D + 0.25 % of Triton X100 + 3% milk powder (without fat) + 0,25 % BSA for 3 hours at 20°C by stirring.
Solutions to be prepared
cacodylate 0.1 M, sodium metabisulfite 10g/l pH 6.2*
(Boer-fixation) 15 ml aqueous saturated picric acid, 5 ml glutaraldehyde (25%), 0.1 ml glacial acetic acid
4% paraformaldehyde in Millonigs-phospate buffer (pH 7.3-7.4, 1g NaCl, 2.9 g Na2HPO42H2O, 0.524g NaH2PO4H2O and 8 g paraformaldehyde were filled up to 200 ml with ddH2O)
0.05 M Tris, sodium metabisulfite 8.5 g/l pH 7.5*
0.05 M Tris, NaCl 8.5 g/l pH 7.5*
*Adjust pH with NaOH or HCl if necessary
Tris solution can be replaced by a 0.01 M phosphate solution.
Loesel et al. (2011) Neuroarchitecture of the arcuate body in the brain of the spider Cupiennius salei (Araneae, Chelicerata) revealed by allatostatin-, proctolin-, and CCAP-immunocytochemistry and its evolutionary implications. Arthropod Struct Dev. 40:210.
Clark et al. (2006) Proctolin-like immunoreactivity in the central and peripheral nervous system of the locust, Locusta migratoria. Peptides 27:549.
Agricola et al. (1985) The distribution of a proctolin-like immunoreactive material in the terminal ganglion of the cockroach Periplaneta americana. Cell Tissue Res. 239:203.
Eckert et al. (1981) Immunocytochemical identification of proctolin-like immunoreactivity in the terminal ganglion and hindgut of the cockroach Periplaneta americana. Cell Tissue Res. 217:633.