A minimal concentration of ASW was used which was shown to elicit Na K ATPase shift . n 5 larva for each experimental group. Each experiment was performed in triplicate. Antibody Production CA9 chicken antibody was generated by Aves Labs, Inc. against the An. gambiae BSA conjugated peptide: KEPIEVSHEQLELFREMRC and was affinity purified using the immunogen peptide . Na K ATPase monoclonal antibodies ?5 that had been raised against the ? subunit of avian Na K ATPase in mice were obtained from the Developmental Studies Hybridoma Bank in the form of hybridoma tissue culture supernatant . A polyclonal V ATPase antiserum raised against the B subunit of the V type H ATPase of Culex quinquefasciatus was obtained from Professor Sarjeet Gill at the University of California, Riverside, USA. Immunolocalization Paraffin sectioned preparations: Primary fixation was achieved by injection into the hemocoel of a 4% formaldehyde solution diluted from ultrapure 16% formaldehyde with Tris buffered saline , and larvae were immersed in 4% formaldehyde overnight at 4 C. Larvae were transferred to Carnoy?s solution for 90 minutes on ice and washed twice with 100% ethanol for 30 minutes each.
Sirolimus Larvae were then cleared with aniline:methylsalicylate overnight, followed by 100% methylsalicylate overnight and embedded in paraffin. Sections were cut six microns thick using a microtome and mounted on gelatin coated slides. Sections were deparaffinized through successive 5 minute incubations in 100% xylene and xylene:ethanol , rehydrated through a series of 5 minute washes of graded ethanols: 100% , 95% , 80%, 70%, 50%, and finally washed three times in TBS. The slides were then blocked in pre incubation buffer for 1 2 hours at room temperature and incubated with primary antibodies to V ATPase and CA9 at a dilution of 1:1000 and Na K ATPase at a dilution of 1:10 in pre inc for 1 hour at 37 C. The slides were washed three times in TBS and incubated with secondary antibodies at a dilution of 1:250 in pre inc for 1 hour at 37 C. The slides were again rinsed three times in TBS and mounted in 60% glycerol in TBS with phenylenediamine to diminish fluorescence quenching.
Whole mount antibody localization was performed to visually distinguish the types of recta: those of freshwater culicines, saline tolerant culicines and freshwater and saline tolerant anophelines, and therefore antibodies were used based on their ability to differentiate one rectal region from another in each species. Whole mount preparations: Larvae were dissected to separate the gut MK-2866 selleck chemicals from the rest of the larva. The dissected tissue was fixed in a 1:1 solution of hemolymph substitute solution and 4% formaldehyde overnight at 4 C. The tissue was then washed twice with TBS for 30 minutes each at room temperature and incubated in pre inc for 1 2 hours at room temperature.