The neurohormone Crustacean Hyperglycaemic Hormone (CHH) is the major member of the CHH-superfamily and controls the osmoregulation, moulting and reproduction in decapod crustaceans.
In some insects a neurohormone occurs, which is closely related to CHH; the Ion Transport Peptide (ITP).
In locusts it is a chloride transport-stimulating and acid secretion-inhibiting hormone and controls (together with other peptide hormones) the final modification of the primary urine by ion, solute and water reabsorptive processes primarily in the hindgut. As ITP is well known in locusts, fruit flies and moths, we want to show its existance in another important model organism for the neuropeptide research: the cockroach Leucophaea maderae (L.maderae).
To localise ITP and the neurosecretory cells, which produce/release ITP, in the brain tissue of L.maderae, we use two indirect immunohistochemical techniques: immunofluorescence staining and peroxidase-antiperoxidase staining. The basic principle of these methods are predicated on the process of detecting antigens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. Because of the similarity to CHH, we can use an antiCHH antibody to stain ITP in the tissue.
To identify ITP in the brain of L.maderae, we conduct a reversed-phase high-performance liquid chromatography (HPLC) and a following direct enzyme-linked immunosorbent assay (ELISA) of 10 retrocerebral complexes.
Table of Contents
Abstract
1. Introduction
2. Materials and Methods
2.1. Leucophaea maderae
2.2. Dissection of Leucophaea maderae
2.2.1. Dissection
2.2.2. Fixation
2.3. Cryostat Microtome Sectioning
2.3.1. Coating the Slides with Poly-L-Lysine
2.3.2. Preparing and Embedding of the Brains
2.3.3. Sectioning
2.4. Vibratome Sectioning
2.4.1 Embedding
2.4.2. Sectioning
2.5. Immunohistochemistry: Immunofluorescence Staining
2.5.1. Antibody-labelling
2.5.2. Mounting
2.6. Immunohistochemistry: Peroxidase-Antiperoxidase Staining
2.6.1. Antibody-labelling and Peroxidase-Antiperoxidase-Complex
2.6.2. Diaminobenzidine-reaction
2.7. Permanent Preparations
2.7.1. Coating the Slides with chromalum-gelatin
2.7.2. Placing sections on coated slides
2.7.3. Dehydration and Mounting
2.8. High-Performance Liquid Chromatography (HPLC)
2.8.1. Dissection for HPLC
2.8.2. Preparation for HPLC: Acidic Extraction
2.8.3. HPLC
2.9. Enzyme-Linked Immunosorbent Assay (ELISA)
2.9.1. Antigen loading
2.9.2. Primary antiserum loading
2.9.3. Goat Anti Rabbit Alkaline Phosphatase Conjugate
2.9.4. Substrate solution loading and photometric measurements
3. Results
3.1. Immunofluorescence Staining
3.1.1. Immunofluorescence stained whole mount preparations
3.1.2. Immunofluorescence stained cryostat sections
3.2. Peroxidase-antiperoxidase Staining
3.3. HPLC and ELISA
3.3.1. HPLC
3.3.2. ELISA
4. Conclusions and Perspectives
5. List of Abbrevations
6. Recipes
7. Principles
7.1. Immunohistochemistry:
7.2. Fluorescence
7.3. Reversed-phase High-performance liquid chromatography
7.4. Enzyme-Linked Immunosorbent Assay
8. Protocols
8.1. Immunofluorescence staining/Antibody labelling Reagent Time
8.2. Peroxidase-Antiperoxidase Reagent Time
9. Other
10. Further Results
11. References
Abstract
The neurohormone Crustacean Hyperglycaemic Hormone (CHH) is the major member of the CHH-superfamily and controls the osmoregulation, moulting and reproduction in decapod crustaceans.
In some insects a neurohormone occurs, which is closely related to CHH; the Ion Transport Peptide (ITP).
In locusts it is a chloride transport-stimulating and acid secretion-inhibiting hormone and controls (together with other peptide hormones) the final modification of the primary urine by ion, solute and water reabsorptive processes primarily in the hindgut. As ITP is well known in locusts, fruit flies and moths, we want to show its existance in another important model organism for the neuropeptide research: the cockroach Leucophaea maderae (L.maderae).
To localise ITP and the neurosecretory cells, which produce/release ITP, in the brain tissue of L.maderae, we use two indirect immunohistochemical techniques: immunofluorescence staining and peroxidase-antiperoxidase staining. The basic principle of these methods are predicated on the process of detecting antigens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. Because of the similarity to CHH, we can use an antiCHH antibody to stain ITP in the tissue.
To identify ITP in the brain of L.maderae, we conduct a reversed-phase high-performance liquid chromatography (HPLC) and a following direct enzyme-linked immunosorbent assay (ELISA) of 10 retrocerebral complexes.
Our work has shown that there is ITP in the brain of the cockroach L.maderae. We were not only able to demonstrate that ITP appears in the retrocerebral complexes (by HPLC and ELISA), but we also showed where ITP is located in the brain and the surrounding periphery (by the immunohistochemical methods).
We found a lot of CHH-immunoreactive structures and cells in the protocerebrum, superior lateral protocerebrum, retrocerebral complex, around the sub-oesophageal ganglion and muscle cells.
Also, our results maybe give some hints about the function of ITP in the cockroach L.maderae.
1. Introduction
Neurohormones are a group of substances produced by specialized nerve cells, the neurosecretory cells, that are structurally similar to nervous rather than endocrine system constituents. The neurohormones pass along axons and are released into the blood- or haemolymph stream at special regions called neurohemal organs. Neurohormones thus constitute a linkage between sensory stimuli and chemical responses (Encyclopaedia Britannica article: Neurohormone).
In crustaceans, the crustacean hyperglycaemic hormone (CHH) is a neurohormone and the major member of the CHH-superfamily type-I. It has several functions such as controlling osmoregulation, moulting and reproduction in decapod crustaceans (Webster et al, 2012). Closely related to CHH is the ion transport peptide (ITP), occurring in insects. CHH and ITP have many characteristic features in common: 1) three disulphide bonds, 2) normally a length of 72-73 amino acids, 3) C-terminal amidation, 4) presence of aromatic amino acids in postition 3 of the N-terminal putative a-helix and 5) same core structure of the first 40 or 41 amino acids (Dircksen, 2008, Webster et al, 2012).
ITP is one of the antidiuretic factors found first in locusts and later in other insects (Dircksen et al, 2008). In locusts, it is a chloride transport-stimulating and acid secretion-inhibiting hormone and controls (together with other peptide hormones) the final modification of the primary urine by ion, solute and water reabsorptive processes primarily in the hindgut. It is also discussed to have an ecdysis-related function in moths (Dircksen, 2008).
In locusts, moth and fruit flies there are two types of ITPs: ITP and the slightly longer isoform ITPL. Both arise by alternative splicing from single ITP genes, that are similar in several insects and crustaceans.
Four principal neuron types, which contain/release ITP/ITPL, are localisated in the same insects as mentioned above in 1) neurosecretory cells of the pars lateralis/retrocerebral complex (usually one splice form only), 2) interneurons (either one of the splice forms), 3) hindgut-innervating abdominal ITP neurons (Drosophila only) and 4) intrinsic, putative sensory neurosecretory cells in peripheral neurohaemal perisympathetic/perivisceral organs or transverse nerves most likely containing ITPLs (Dircksen, 2008).
As ITP is well known in locusts, fruit flies and moths, we wanted to show its existance in another important model organism for the neuropeptide research: the cockroach Leucophaea maderae (L.maderae).
Like all insects, L.maderae looses water through the soft cuticule, and it lives in areas where it is often exposed to water shortage. Therefore, L.maderae is dependent upon saving and/or regaining of water and needs a hormone such as ITP. The primary aim of this study is to localise and identify the ITP-releasing neurons in the brain of L.maderae.
A well established method for the specific localisation of different substances in tissue sections is immunohistochemistry. It refers to the process of detecting antigens (e.g., proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues (Wikipedia article: Immunohistochemistry). Several procedures are used to obtain this type of information, based on high-affinity interactions between macromolecules, in the end leading to insoluble colored (or electron-dense) compounds, and so the localisation of specific substances with the help of light, and/or electron microscopy is possible (Junqueria et Carneiro, 2003).
For the localization of the ITP releasing neurons in the brain and periphery of the cockroach L.maderae, we applied two different indirect immunohistochemical methods. The first is the immunofluorescence staining and the second the peroxidase-antiperoxidase (PAP) staining method. As a primary antibody for the stainings we used rabbit antiCHH antibodies, which are polyclonal. Given that CHH has a very similar structure to ITP, an anti-crayfish CHH antibody can likely be used to stain cross-reactive ITP in the neurons of the L.maderae brain. The specificity of the primary antibody is however not high enough to distinguish between ITP and ITPL. Thus, in these experiments both splice forms are stained.
For the identification of ITP and ITPL in the nervous system of L.maderae we applied a high-performance liquid chromatography (HPLC) separation of extracts, followed by a direct enzyme-linked immunosorbent assay (ELISA) of the retrocerebral complex (corpora cardiaca and corpus allatum). We use the retrocerebral complex for the HPLC & ELISA, because many neurohaemal organ-like structures have been found within insects previously via immunoreactivity to CHH antibodies (Lucas, 2006).
2. Materials and Methods
2.1. Leucophaea maderae
During all experiments the cockroach Leucophaea maderae (L.maderae) was used as a model organism.
Adult cockroaches were taken from laboratory colonies kept under crowded conditions (at least 50 animals per 0,175m²) at room temperature in a fume hood. They were reared under a 12:12 h light-dark photoperiod. The animals were fed every 2-3 days with omnivore food, like meat, vegetables (e.g. eggplants, salad), fruits (e.g. apples, oranges), cheese and bread.
2.2. Dissection of Leucophaea maderae
2.2.1. Dissection
The animals were caught (one by one) using large forceps and put directly into a killing jar containing ethyl acetate vapours. The vapours killed the insects quickly without destroying them. After the cockroaches had died, the head was cut of with dissection scissors. The head was placed into a dissection bowl filled with Sylgard® plastics and Leucophaea-maderae ringer-solution (recipe in appendix). The head was dissected under a stereo-microscope and with the help of a pair of forceps and dissection scissors. The whole brain with the optic lobes, corpora cardiaca, corpora allata, sub-oesophageal ganglion, frontal ganglion and some peripheral nerves was dissected. The brain was largely freed from other tissues such as trachea, cuticule, muscles, mandibles, fat body etc.
2.2.2. Fixation
The fully dissected brains were fixed immediately by submersion. Therefore, the brains were placed on a piece of filter-paper, quickly blotted to near dryness and then put upside down into a lymph vessel filled with Zamboni‘s fixative (recipe in appendix). Because of being reasonably attached to the filter paper, the brains kept their original shape and connections. The active components in Zamboni‘s fixative are picric acid, which precipitates and derivatises proteins, and formaldehyde, which cross-links the proteins in the tissue, thus preserving the tissues from decay (Wikipedia article: Fixation (histology)). The brains were incubated in the fixative overnight at room temperature.
2.3. Cryostat Microtome Sectioning
2.3.1. Coating the Slides with Poly-L-Lysine
The Poly-L-Lysine solution (0.1% w/v, in water, containing Thimerosal, 0,01%, added as preservative, Sigma-Aldrich, Germany) was diluted to a final concentration to 0,01% with deionized water. The clean slides were placed in the diluted solution for 5 minutes. Afterwards they were drained and left to dry at room temperature overnight.
By the use of Poly-L-Lysine for the coating of the slides, the sections adhered more strongly to the glass surface.
2.3.2. Preparing and Embedding of the Brains
After the fixation of the dissected brains, the fixative and the filter-paper were removed, and the preparations were rinsed 3 times for 10 minutes in 0.1M phosphate buffer (0.1M PB) at room temperature. Subsequently, the preparations were incubated in a solution series with increasing sucrose concentrations, to introduce a freezing protectant before cryostat microtome sectioning. They were placed in each of the following sucrose-solutions for one and a half hour at room temperature under gently shaking: 5% sucrose, 10% sucrose and 20% sucrose in 0.1M PB. Each solution contained 0.02% sodium-azide.
Thereafter the preparations were embedded in freezing medium (Tissue Tek O.C.T. TM Compound): A drop of the freezing medium was placed on the metal tissue holder of the cryostat microtome and frozen in the cryostat microtome at -27°C. In a next step, again a small amount of freezing medium was put on the covered metal tissue holder and the sucrose-infiltrated brain was placed into it. The sample was then immediately inserted into the cryostat microtome at -27°C to allow for further freezing and equilibration.
2.3.3. Sectioning
For the sectioning, the cryostat microtome Leica CM 1850 UV, Germany, was used. The freezing chamber was cooled down on -27°C and the sections had a thickness of 20µm. Consecutive sections were collected on Poly-L-Lysine slides, with the help of a small paint brush. After sectioning, the slides were left in the freezing chamber of the cryostat microtome at -27°C overnight.
2.4. Vibratome Sectioning
2.4.1 Embedding
After the brains were fixed in Zamboni’s fixative overnight, the fixative and the filter-paper were removed. Subsequent, the brains were washed 4 times for 10 minutes with 0.1M PB under gentle shaking and incubated for 10 seconds at room temperature in a 5% gelatin/0.1M PB solution. The gelatin/albumin embedding medium (recipe in appendix) was melted by heating slightly in a water bath to about 45-50°C. A teflon form (1x1x1.5cm3) was filled with the medium, the brain was placed into it and orientated immediately. The embedding medium was then left to harden by keeping it at 4°C for 30 minutes. Subsequently, the form was placed into a 3.7%-formalin solution for 1h at 4°C to prefix the embedding medium. Then the blocks with the embedded brains were taken out of the form and fixed further in the 3.7%-formalin solution overnight at 4°C in a tightly stoppered container. Blocks were briefly washed and kept in 0.1M PB at 4°C.
2.4.2. Sectioning
The sectioning was done with a vibrating blade microtome (Vibratome Series 1000 Sectioning System, Bachofer, Germany). While being sectioned, the preparations were immersed in a 0.1M phosphate buffered saline (0.1M PBS): The embedded preparations were cut with a razor blade in a trapezoidal shape and then glued with instant adhesive to a metal block. The block was placed and clamped into the holder of the vibratome. The preparations were sectioned at a speed of 2 and an amplitude of 4.5; every section had a thickness of 50µm. To store and later wash the sections, they were kept free-floating in 0.1M PB in sterile 24-well-plates.
2.5. Immunohistochemistry: Immunofluorescence Staining
Whole mount preparations or frozen sectiones were labelled using immunofluorescence staining. The same protocol was used for both, but the whole mount preparations were stained and washed in watch glasses and the frozen sections directly on the Poly-L-Lysine-coated slides.
2.5.1. Antibody-labelling
To prepare whole mounts and section of brains for the labelling with the primary antibody, samples were washed 3 times for 10 minutes with 0.1M PB and then 3 times for 10 minutes with Tris-buffered saline, containing 0.1% Triton X-100 (0.1M TBTX), to remove most of the lipids from the tissue. Subsequently, they were incubated overnight at room temperature in the primary antibody solution. For the whole mount preparations, the primary antibody antiOrconectes CHH T5 B4/70 (1:5000) in 0.1M TBTX, containing 0.02% sodium-azide, was used. For the frozen sections, the primary antibody anti-OCHH T5 B/4 (1:8000) in 0.1M TBTX, containing 0.02% sodium-azide, was used. The primary antibody targets the antigen ITP in the tissue.
After the incubation, the primary antibody solution was removed, and the brains were washed 3 times for 15 minutes with 0.1M TBTX and afterwards incubated for 2 hours at room temperature in the secondary antibody solution. For the whole mount preparations, the secondary antibody GAR-FITC (Goat anti Rabbit IgG (whole molecule) Fluorescein Isothiocyanate (FITC) Conjugate, Sigma-Aldrich, Germany) 1:100 in 0.1M TBTX, containing 0.02% sodium-azide, was used and for the frozen sections the secondary antibody GAR-Cy3 (Goat anti Rabbit, Cyanin 3) 1:1000 in 0.1M TBTX, containing 0.02% sodium-azide. The secondary antibody, which is loaded with a fluorescence marker (FITC or Cy3), binds specifically to the primary rabbit antibody. Thereafter the preparations were washed 3 times for 10 minutes with 0.1M TBTX.
The detailed protocol for the Antibody-labelling/Immunofluorescence staining can be found in the appendix (6.3.1).
2.5.2. Mounting
To mount the whole mount preparations, they were placed with 0.1M PB into the concave well in the middle of an hour glass slide; The buffer was then sucked off. A small amount (500µl) of a solution containing 80% glycerol (Merck) and 50mg/ml 1,4-Diazabicyclo[2.2.2]Octane (DABCO, Triethylenediamine, Sigma-Aldrich, Germany) was then pipetted onto the whole mounts or frozen section on the slides. Cover slips (size: 24 x 50mm2) were finally placed on the slides.
The preparations were then ready for being viewed under the fluorescence microscope (Zeiss Axioplan 2 or Leitz Aristoplan).
2.6. Immunohistochemistry: Peroxidase-Antiperoxidase Staining
2.6.1. Antibody-labelling and Peroxidase-Antiperoxidase-Complex
After the Vibratome sectioning, the free-floating sections were washed 3 times for 10 minutes with 0.1M TBTX and then preincubated in 2% NGS in 0.1M TBTX, containing 0.02% sodium-azide, for 1 hour at room temperature. Subsequently, they were incubated in a primary antibody solution at room temperature overnight continous gently shaking. The primary antibody anti-OCHH T5 B/4 (1:8000) in 0.5M TBTX, containing 1% NGS and 0.02% sodium azide, was used.
Afterwards, the sections were rinsed 3 times for 10 minutes in 0.1M TBTX. The second antibody labelling was done with GAR (Goat anti Rabbit IgG without any marker, ICN ImmunoBiologicals) (1:100) in 0.1M TBTX, containing 1% NGS and 0.02% sodium-azide, for 1 hour at room temperature under gentle shaking. The sections were washed 3 times for 10 minutes with 0.1M TBTX after the second antibody labelling. Thereafter, the sections were incubated in rabbit PAP (1:300) in 0.5M TBTX, containing 1% NGS, for 1 hour at room temperature while gently shaking. The rabbit-PAP-comples, consisting of three peroxidase and two anti-peroxidase molecules, binds to the secondary antibody GAR (Sternberger, 1974). Subsequently, they were washed 3 times for 10 minutes with 0.1M TBTX and additionally one time for 10 minutes with 0.1M PB.
2.6.2. Diaminobenzidine-reaction
Diaminobenzidine(DAB) solutions were always freshly made before use. 10 mg DAB powder (3,3‘-Diaminobenzidine tetrahydrochloride hydrate, >96%, Sigma-Aldrich, Germany) was first diluted in 500µl distilled water and then added to 30ml 0.1M PB, containing 10µl H₂O₂ (from 30% stock). The DAB solution was poured onto the PAP-labelled sections. DAB is oxidized in the presence of peroxidase and a brown precipitate is produced consequently, which can be seen under a light (or electron) microscope (Junqueira et Carnerio, 2003). The reaction time was 10-45 minutes under visual control with a stereo-microscope. As soon as the sections showed a brown background staining, the reaction was stopped. Thereafter, the sections were rinsed 3 times for 2 minutes in distilled water. Finally, they were washed once in 0.1M PB and also kept in 0.1M PB at 4°C.
The detailed protocol for the PAP method is given in the appendix.
2.7. Permanent Preparations
2.7.1. Coating the Slides with chromalum-gelatin
The slides were cleaned in a acetone/ethanol(1:1) solution overnight. The chromalum-gelatin solution (recipe see appendix) was always freshly prepared. The slides were put one by one put for 10 seconds into the chromalum-gelatin solution, slowly removed and afterwards leaned against a vessel to drain. The slides were then placed in a vessel and dried overnight at 60°C.
2.7.2. Placing sections on coated slides
To place sections on a chromalum-gelatin coated slide, a small amount of 0.1M PB was pipetted on the slide. Consecutive sections were then positioned one by one on the slide and left to dry at room temperature. A small amount of a 2% gelatin-solution was finally placed on top of the sections in order to avoid folding up during drying, which later proceeded at room temperature overnight.
2.7.3. Dehydration and Mounting
For permanent preparations, the sections were dehydrated in an increasing alcohol series and cleared in xylene. All solutions in this procedure were provided in 250ml cuvettes, and all incubations were done at room temperature. The slides with the sections were incubated for 10 minutes in 0.1M PB and then for 3 minutes in distilled water. Accordingly, the slides were incubated for 3 minutes in each of the following alcohol concentrations: 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 100%. Afterwards, the slides were cleared 3 times for 3 minutes in xylene (3 different vessels).
Subsequently, a small amount (1-2ml) of DEPEX (DPX mountant for microscopy (contains xylene (mixture of isomers), dibutyl phthalate, BDH laboratory supplies, England) was placed on each slide and a cover slip on top. They were then left to dry at room temperature overnight.
The permanent preparations were then viewed using bright-field optics under a Leitz Aristoplan microscope.
2.8. High-Performance Liquid Chromatography (HPLC)
2.8.1. Dissection for HPLC
Animals were sacrificed as described under 3.2.1 Dissection. For high-performance liquid chromatography (HPLC) separation only the retrocerebral complexes (RCC) (corpora cardiaca and corpora allata) were dissected. These dissections were quickly done under a stereo-microscope in a dissection bowl filled with Leucophaea-maderae ringer-solution. Finished preparations were collected on the bottom of dry ice-chilled 1,5 ml Eppendorf tubes and finally kept at -80°C.
2.8.2. Preparation for HPLC: Acidic Extraction
At first, 500µl of 2M acetic acid were pipetted onto 10 L.maderae RCCs in the Eppendorf tube (1,5ml). This solution was treated with a ultrasonic disruptor 3 times for 30 seconds on ice. Afterwards, it was centrifuged for 10 minutes at 16.000g at 4°C. The supernatant was pipetted out and saved in a new Eppendorf tube (1,5ml). Again 500µl of 2M acetic acid was poured on the pellett, and the new solution was treated with the ultrasonic disruptor 3 times for 30 seconds on ice. Subsequently, the solution was centrifuged a second time for 10 minutes at 16.000g at 4°C. The supernatant was transferred together with the supernatant from the first centrifugation. Then, the solution was dried in a vacuum centrifuge (SpeedVac SC100, Savant coupled to a Freeze Dryer 4.5 Labconco; Pfeiffer Vacuum Pump) for ca. 1 hour at -50°C. Afterwards, the fractions were resuspended with 200µl 2M acetic acid and again centrifuged for 10 minutes at 16.000g at 4°C before injection into the HPLC.
2.8.3. HPLC
The reversed-phase HPLC fractionation of the acidic extracts was performed on a Waters HPLC system with a U6K injector, a model 600E controller, and a model 486 ultraviolet detector (set to 210nm). A Waters Millennium 2010 chromatography manager was used for data acquistion and processing. After 2 minutes of initial conditions, a linear gradient was applied from 18% to 48% acetonitrile containing 0.1% Trifluoroacetic acid (TFA) for 60 minutes (MeCN-TFA; 0,5% MeCN/minute) followed by a final 10-minute gradient to 60% MeCN-TFA at a flow rate of 0.9 ml/minute. During chromatography on a µ-Bondapak phenyl column (4,6 mm x 250 mm; Waters; with a guard column) peak fractions were collected manually (Dircksen et al. 2008).
Afterwards, the fractions were dried in the vacuum centrifuge for 1 hour at -50°C and then kept at -20°C.
2.9. Enzyme-Linked Immunosorbent Assay (ELISA)
2.9.1. Antigen loading
At first, 100µl of 60% acetonitrile was pipetted on every dry fraction, that was then vortexed for 10 seconds, put in an ultrasonic bath (Sonorex RK 255 S, Bandelin) for 15 seconds and then vortexed again for 10 seconds.
From every HPLC fraction and a blank sample, 10µl were loaded in 2 wells each of a sterile 96-well-plate (pipetting scheme in appendix). The plate was then dried under vacuum for half an hour at -50°C.
Subsequently, 100µl of 0.1M sodium-carbonate buffer (pH 9.6) were added to every well, and the plate was then incubated for 2 hours at 37°C in a humid chamber.
2.9.2. Primary antiserum loading
The sodium-phosphate buffer was chucked out from the 96-well-plate and all wells washed 3 times for 5 minutes with 150µl PBS-Tween-azide (see appendix for buffer details) per well, while shaking at 450rpm. Subsequently, 100µl of the primary antiserum solution (antiOCHH T5 B/4 (1:8000) in PBS-Tween-azide, containing 1% NGS) was pipetted in each well. The samples were incubated overnight at 4°C in the primary antiserum.
2.9.3. Goat Anti Rabbit Alkaline Phosphatase Conjugate
The primary antiserum was chucked out from the 96-well-plate, and the samples in the wells were again washed 3 times for 5 minutes with 150µl PBS-Tween-azide per well under rapid shaking (shaker agitation 450 rpm).
Then 100µl of 1% goat anti rabbit alkaline phosphatase (GAR-AP) in PBS-Tween-azide, containing 1% NGS, was pipetted in each well, and incubated for 1,5 hours at 37°C in a humid chamber.
2.9.4. Substrate solution loading and photometric measurements
The GAR-AP solution was chucked out from the plate, and the samples were washed again 3 times for 5 minutes with 150µl PBS-Tween (without azide), under agitation of 450rpm. Afterwards, 100µl of the substrate solution consisting of 0.1% para-nitrophenyl phosphate (4-Nitrophenylphosphate Disodium salt, Hexahydrate, >97% (NT), Fluka Biochemika, Germany) in Na-carbonate buffer pH 9.6) were pipetted into each well. After 15, 30 and 45 minutes, the absorption of the samples were measured with an ELISA reader (Titertek Multiskan Plus MK 2).
3. Results
3.1. Immunofluorescence Staining
3.1.1. Immunofluorescence stained whole mount preparations
Abbildung in dieser Leseprobe nicht enthalten
Fig. 1. Immunohistochemical localisation of the Ion transport peptide in whole mount preparations of the brain of Leucophaea maderae. (a-e) The preparations are stained with the primary antibody antiCHH and the secondary antibody GAR-FITC. (a-b) In the protocerebrum of the brain of L.maderae two cell groups with a immunoreactivity to the antiCHH antibody are shown. They are localisad in the frontal part, one group on each side (arrows). (c-e) The sub-oesophageal ganglion (SOG) of the L.maderae brain is shown. Many structures, which are immunoreactive to the antiCHH antibody, occur in the periphery around the SOG nerves and nerve roots. (e) Fibre networks in the corpus allatum (CA), heavily stained by the antiCHH antibody, can be seen above the SOG.
Two bilaterally symmetrical cell groups occur in the frontal part of the protocerebrum of the brain, which showed an immunoreactivity to the antiOCHH antibody (Fig. 1a and 1b, marked by the arrows).
Furthermore, we discovered a lot of fibres in the periphery around the sub-oesophageal ganglion, which were heavily stained by the antiCHH antibody (fig. 1c and 1d). The retrocerebral complex also showed a strong immunreactivity to the antiCHH antibody, especially the corpus allatum showed abundant fibres and terminals of an intense fluorescence ocurred around the glandular cells (Fig. 1e). Comparatively less abundant fibres and terminals were found in the corpora cardiaca.
Frequently asked questions
What is the purpose of this document?
This document provides a comprehensive language preview, including the title, table of contents, objectives and key themes, chapter summaries, and key words related to a research study. It serves as an introduction and overview of the research.
What is the main topic of the research described in this document?
The research focuses on the neurohormone Ion Transport Peptide (ITP) in the cockroach Leucophaea maderae (L.maderae). It aims to localize and identify ITP-releasing neurons in the brain of this cockroach, comparing it to the well-known Crustacean Hyperglycaemic Hormone (CHH) found in crustaceans and ITP in locusts, fruit flies, and moths.
What methods were used in this research?
The research employed a variety of methods, including:
- Dissection of L.maderae brains
- Cryostat microtome sectioning
- Vibratome sectioning
- Immunohistochemistry: Immunofluorescence staining
- Immunohistochemistry: Peroxidase-Antiperoxidase (PAP) staining
- High-Performance Liquid Chromatography (HPLC)
- Enzyme-Linked Immunosorbent Assay (ELISA)
What is the purpose of using immunohistochemistry in this research?
Immunohistochemistry, specifically immunofluorescence staining and peroxidase-antiperoxidase staining, was used to localize ITP and the neurosecretory cells that produce/release ITP within the brain tissue of L.maderae. AntiCHH antibodies were used to stain ITP due to its structural similarity to CHH.
Why was HPLC and ELISA used in this research?
HPLC (High-Performance Liquid Chromatography) was used to separate extracts from retrocerebral complexes of L.maderae. ELISA (Enzyme-Linked Immunosorbent Assay) was then used to identify and quantify ITP in the separated fractions, confirming its presence in the cockroach brain.
What are the key findings of the research?
The research demonstrated the presence of ITP in the brain of the cockroach L.maderae. ITP was identified in the retrocerebral complexes through HPLC and ELISA, and its location within the brain and surrounding periphery was mapped using immunohistochemical methods. CHH-immunoreactive structures and cells were found in various brain regions including the protocerebrum, retrocerebral complex, and around the sub-oesophageal ganglion.
What is the significance of finding ITP in Leucophaea maderae?
The discovery of ITP in L.maderae, an important model organism for neuropeptide research, expands our understanding of the distribution and potential function of this hormone in insects. Given L.maderae's susceptibility to water loss, the presence of ITP suggests a role in water regulation.
What is Zamboni's fixative, and why was it used?
Zamboni's fixative is a solution used to preserve tissue samples for microscopic examination. Its active components, picric acid and formaldehyde, precipitate proteins and cross-link them within the tissue, preventing decay and maintaining the brain's original shape during dissection and processing.
What does this document tell me about the culture of cockroaches?
The cockroaches (Leucophaea maderae) were kept under specific laboratory conditions: crowded (at least 50 animals per 0,175m²), at room temperature in a fume hood, under a 12:12 h light-dark photoperiod, and fed every 2-3 days with omnivore food like meat, vegetables, fruits, cheese, and bread.
What are the recipes and protocols mentioned?
The document mentions recipes for Leucophaea-maderae ringer-solution, Zamboni‘s fixative, gelatin/albumin embedding medium and chromalum-gelatin solution. It also refers to detailed protocols for Antibody-labelling/Immunofluorescence staining and the PAP (Peroxidase-Antiperoxidase) method, which can be found in the appendix.
- Arbeit zitieren
- Janna Witt (Autor:in), 2012, Localisation and Identification of Ion Transport Peptide in the Brain of the Cockroach Leucophaea maderae, München, GRIN Verlag, https://www.grin.com/document/1322201
