We are grateful to Chau-Ti Ting (Fly Core in Taiwan) for providing the monoclonal 4D9 antibody, Technology Commons (TechComm) of the College of Life Science NTU for confocal microscopy, Hsiao-Ling Lu for proofreading the manuscript, and Chen-yo Chung for helping filming. CC particularly thanks Charles E. Cook for providing strategic suggestions and for critical editing of the manuscript. This work was supported by the Ministry of Science and Technology (101-2313-B-002-059-MY3 and 104-2313-B-002-022-MY3 for GWL and CC), and the National Taiwan University (NTU-CESRP 101R4602D3 for CC; 103R4000 for GWL).

1. Culture of Aphids
NOTE: The laboratory strain of the parthenogenetic viviparous pea aphid A. pisum was originally collected in the central Taiwan and has been reared on host plants (the garden pea Pisum sativum or broad bean Vicia faba) under long-day photoperiod for more than 300 generations (one generation: ~10 days).
<ol>
	<li>Germination of Seeds &#160; &#160; &#160;
	<ol>
		<li>Soak the seeds of host plants in tap water for 3-5 days at RT. Refill with fresh water once ...

<ol>
	<li>Blackman, R. L., Minks, A. K., Harrewijn, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproduction,+cytogenetics+and+development.">Reproduction, cytogenetics and development.</a> Aphids: their biology, natural enemies. , 163-195 (1987).</li><li>Davis, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclical+parthenogenesis+and+viviparity+in+aphids+as+evolutionary+novelties.">Cyclical parthenogenesis and viviparity in aphids as evolutionary novelties.</a> J. Exp. Zool. 318, 448-459 (2012).</li><li>The International Aphid Genomics Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+sequence+of+the+pea+aphid+Acyrthosiphon+pisum.">Genome sequence of the pea aphid Acyrthosiphon pisum.</a> PLoS Biol. 8 (2), e1000313 (2010).</li><li>Abzhanov, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+we+there+yet?+Tracking+the+development+of+new+model+systems.">Are we there yet? Tracking the development of new model systems.</a> Trends Genet. 24 (7), 353-360 (2008).</li><li>Chang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apvasa+marks+germ-cell+migration+in+the+parthenogenetic+pea+aphid+Acyrthosiphon+pisum+(Hemiptera:+Aphidoidea)).">Apvasa marks germ-cell migration in the parthenogenetic pea aphid Acyrthosiphon pisum (Hemiptera: Aphidoidea)).</a> Dev. Genes Evol. 217 (4), 275-287 (2007).</li><li>Chang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+identification+of+gene+transcripts+in+aphids:+Protocols+and+evaluation+of+probe+accessibility.">Whole-mount identification of gene transcripts in aphids: Protocols and evaluation of probe accessibility.</a> Arch. Insect Biochem. 68 (4), 186-196 (2008).</li><li>Chung, C. Y., Cook, C. E., Lin, G. W., Huang, T. Y., Chang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+protocols+for+whole-mount+fluorescent+in+situ+hybridization+(FISH)+in+the+pea+aphid+Acyrthosiphon+pisum:+a+comprehensive+survey+and+analysis.">Reliable protocols for whole-mount fluorescent in situ hybridization (FISH) in the pea aphid Acyrthosiphon pisum: a comprehensive survey and analysis.</a> Insect Sci. 21 (3), 265-277 (2014).</li><li>Mutti, N. S., Park, Y., Reese, J. C., Reeck, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+knockdown+of+a+salivary+transcript+leading+to+lethality+in+the+pea+aphid,+Acyrthosiphon+pisum.">RNAi knockdown of a salivary transcript leading to lethality in the pea aphid, Acyrthosiphon pisum.</a> J. Insect Sci. 6, 38 (2006).</li><li>Jaubert-Possamai, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+knockdown+by+RNAi+in+the+pea+aphid+Acyrthosiphon+pisum.">Gene knockdown by RNAi in the pea aphid Acyrthosiphon pisum.</a> BMC Biotechnol. 7, 63 (2007).</li><li>Sapountzis, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insight+into+the+RNA+interference+response+against+cathepsin-L+gene+in+the+pea+aphid,+Acyrthosiphon+pisum:+Molting+or+gut+phenotypes+specifically+induced+by+injection+or+feeding+treatments.">New insight into the RNA interference response against cathepsin-L gene in the pea aphid, Acyrthosiphon pisum: Molting or gut phenotypes specifically induced by injection or feeding treatments.</a> Insect Biochem. Mol. Biol. 51, 20-32 (2014).</li><li>Braendle, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+origin+and+evolution+of+bacteriocytes+in+the+aphid-Buchnera+symbiosis.">Developmental origin and evolution of bacteriocytes in the aphid-Buchnera symbiosis.</a> PLoS Biol. 1 (1), e21 (2003).</li><li>Miura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+parthenogenetic+and+sexual+embryogenesis+of+the+pea+aphid+Acyrthosiphon+pisum+(Hemiptera:+Aphidoidea).">A comparison of parthenogenetic and sexual embryogenesis of the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidoidea).</a> J. Exp. Zool. 295 (1), 59-81 (2003).</li><li>Chang, C., Lee, W. C., Cook, C. E., Lin, G. W., Chang, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ-plasm+specification+and+germline+development+in+the+parthenogenetic+pea+aphid+Acyrthosiphon+pisum:+Vasa+and+Nanos+as+markers.">Germ-plasm specification and germline development in the parthenogenetic pea aphid Acyrthosiphon pisum: Vasa and Nanos as markers.</a> Int. J. Dev. Biol. 50 (4), 413-421 (2006).</li><li>Koshy, A. A., Cabral, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-D+imaging+and+analysis+of+neurons+infected+in+vivo+with+Toxoplasma+gondii.">3-D imaging and analysis of neurons infected in vivo with Toxoplasma gondii.</a> J. Vis. Exp. (94), e52237 (2014).</li><li>Brisson, J. A., Stern, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pea+aphid,+Acyrthosiphon+pisum:+an+emerging+genomic+model+system+for+ecological,+developmental+and+evolutionary+studies.">The pea aphid, Acyrthosiphon pisum: an emerging genomic model system for ecological, developmental and evolutionary studies.</a> Bioessays. 28 (7), 747-755 (2006).</li><li>Tautz, D., Pfeifle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-radioactive+in+situ+hybridization+method+for+the+localization+of+specific+RNAs+in+Drosophila+embryos+reveals+translational+control+of+the+segmentation+gene+hunchback.">A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback.</a> Chromosoma. 98 (2), 81-85 (1989).</li><li>Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-situ+hybridization:+an+improved+whole-mount+method+for+Xenopus+embryos.">In-situ hybridization: an improved whole-mount method for Xenopus embryos.</a> Method. Cell Biol. 36, 685-695 (1991).</li><li>Wolff, C., Sommer, R., Schroder, R., Glaser, G., Tautz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+and+divergent+expression+aspects+of+the+Drosophila+segmentation+gene+hunchback+in+the+short+germ+band+embryo+of+the+flour+beetle+Tribolium.">Conserved and divergent expression aspects of the Drosophila segmentation gene hunchback in the short germ band embryo of the flour beetle Tribolium.</a> Development. 121 (12), 4227-4236 (1995).</li><li>Jowett, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+in+situ+hybridization+techniques+in+zebrafish.">Double in situ hybridization techniques in zebrafish.</a> Methods. 23 (4), 345-358 (2001).</li><li>Lin, G. W., Cook, C. E., Miura, T., Chang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posterior+localization+of+ApVas1+positions+the+preformed+germ+plasm+in+the+sexual+oviparous+pea+aphid+Acyrthosiphon+pisum.">Posterior localization of ApVas1 positions the preformed germ plasm in the sexual oviparous pea aphid Acyrthosiphon pisum.</a> Evodevo. 5, 18 (2014).</li><li>Straus, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+peroxidase+by+methanol+and+by+methanol-nitroferricyanide+for+use+in+immunoperoxidase+procedures.">Inhibition of peroxidase by methanol and by methanol-nitroferricyanide for use in immunoperoxidase procedures.</a> J. Histochem. Cytochem. 19 (11), 682-688 (1971).</li></ol>

We identified optimal conditions critical to successful immunostaining in the pea aphid A. pisum, an emerging model organism for genomic and developmental studies3,15. Optimized conditions for increasing tissue permeability and reducing background staining enhanced the intensity and specificity of signals. They differ from standard protocols for immunostaining in other animal models in the steps for creating pores in cell membranes and blocking non-specific antibody binding.
<p class="jov...

Aphids are hemipteran insects with small (1-10 mm) soft bodies. They feed on plants by sucking phloem sap with piercing mouthparts. Additionally, they rely on an obligate endosymbiotic bacterium, Buchnera aphidicola, to synthesize essential amino acids that are deficient in the phloem sap diet. Aphids have a complex life history that includes parthenogenetic viviparous reproduction during spring and summer long-day photoperiods and sexual oviparous reproduction triggered by short-day photoperiods during which they lay a limited number of overwintering eggs1,2. In spring these eggs hatch to produce the first generation of all-female aphids (fundatrices), following many rounds of parthenogenetic reproduction until autumn. The cyclical parthenogenesis in aphids, where asexual and sexual phases alternate in the annual life cycle, has been regarded as an evolutionary novelty1,2. In the parthenogenetic viviparous aphids, embryogenesis takes place within egg chambers of the ovarian tubules (ovarioles). By contrast, sexual oviparous embryos develop in the fertilized eggs. Apart from reproductive plasticity, aphids can display transgenerational wing polyphenism: in response to overcrowding signals and predator threats, the unwinged asexual females can viviparously produce winged offspring for long-distance migration. Publication of the genome sequence of the pea aphid Acyrthosiphon pisum-the first genome sequence for a basal hemimetabolous insect-allows further exploration of reproductive plasticity, wing polyphenism, and other features including insect-plant interactions, viral vectoring and symbiosis in aphids on a molecular basis3.
In addition to the sequenced genome, tools for characterizing gene expression and function are required for promoting the pea aphid as a mature model organism4. We have described robust protocols of whole-mount in situ hybridization for detecting expression of mRNA in aphid embryos5-7. RNA interference (RNAi) via double-stranded RNA injection and feeding has been used for gene silencing in aphid nymphs and adults, but stable conditions for gene knockdown in the embryos have not yet been reported8-10. Immunostaining, an antibody-based approach that can detect protein expression in samples before and after RNAi knockdown, has been performed on pea aphid embryos11-13. However, increase of tissue permeability and elimination of background staining are as yet unsatisfactory using standard protocols for immunostaining in the asexual viviparous embryos of the pea aphid. For example, we found that penetration of antibody to the tissues decreased in gastrulating embryos (stages 8-10) and that embryos with morphologically identifiable limb buds (stages 13-14) were barely permeable to antibody. In addition, background staining was visualized in the asexual viviparous pea aphid embryos stained using antibody against the germline marker Vasa as well as that against the Engrailed/Invected protein expressed in the embryonic segments12,13. Actually background staining was still clearly visible in embryos stained with the secondary antibody alone.
In order to increase permeability without damaging integrity of aphid tissues, we carefully titrated the concentration of proteinase K and determined optimal conditions for tissue digestion on aphid embryos. In order to avoid non-specific staining in the pea aphid, we searched for compounds that could effectively block embryos and suppress activity of endogenous peroxidase (POD), an enzyme employed for amplifying signals during immunostaining. A blocking reagent provided by a Digoxigenin (DIG)-based buffer set, rather the traditionally used normal goat serum (NGS)/bovine serum albumin (BSA), significantly reduced background staining. Moreover, methanol was found to inhibit the endogenous POD activity more effectively than hydrogen peroxide (H2O2). Details regarding these aphid-specific conditions for immunostaining on embryos will be described in the following sections.

<table>
	<tbody>
		<tr>
			<td>30% hydrogen perxidase (H&#8322;O&#8322;)</td>
			<td>Sigma-Aldrich</td>
			<td>18304</td>
			<td>For bleaching endogenous peroxidase of embryos.</td>
		</tr>
		<tr>
			<td>4&#8217;6-diamidino-2-phenyl-indole dihydrochloride (DAPI)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>For labeling the double-strand DNA. TOXIC. Wear gloves, dispense into small aliquots and feeze to minimize exposure</td>
		</tr>
		<tr>
			<td>4D9 anti-engrailed/invected mouse monoclonal antibody</td>
			<td>Developmental studies hybridoma bank</td>
			<td>AB_528224</td>
			<td>For labeling the developing segments of embryo.</td>
		</tr>
		<tr>
			<td>ApVas1 rabbit polyclonal antibody</td>
			<td>Our laboratory</td>
			<td>N/A</td>
			<td>For labeling the aphid germline specific Vas1 protein in embryos. This antibody was made by our laboratory.&#160;</td>
		</tr>
		<tr>
			<td>Austerlitz Insect pins</td>
			<td>ENTOMORAVIA</td>
			<td>N/A</td>
			<td>For seperating each egg chambers from an ovariole. Size 000, BLACK ENAMELLED.&#160;</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>9048-46-8</td>
			<td>For blocking the non-specific binding of antibodies.</td>
		</tr>
		<tr>
			<td>Camera connted to compound microscope</td>
			<td>Canon</td>
			<td>EOS 5D</td>
			<td>For photographing of aphid embryos.</td>
		</tr>
		<tr>
			<td>Colorimetric 8 cell tray</td>
			<td>Kartell Labware</td>
			<td>357</td>
			<td>For developing the staining signal of aphid embryos. Diameter 95 x 57 mm, cell depth 2 mm.</td>
		</tr>
		<tr>
			<td>Compound microscope with DIC optics</td>
			<td>Leica Microsystems</td>
			<td>DMR</td>
			<td>For photographing of aphid embryo mounted on the slides.</td>
		</tr>
		<tr>
			<td>DIG Wash and Block Buffer Set</td>
			<td>Sigma-Aldrich (Roche)</td>
			<td>1.1586E+10</td>
			<td>For blocking the non-specific binding of antibodies. We diluted the 10X Blocking solution into 1X as blocking reagent.</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Ideal-tek</td>
			<td>N/A</td>
			<td>For dissection of ovaries from aphids. Manufacturer part No 5: 5 SA.&#160;</td>
		</tr>
		<tr>
			<td>Glass dropper</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For transfering ovaries of aphids from the splot plate to tubes. 150 mm of total length and 5 3/4&#34; of tip length.</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td>For clearing of aphid embryos and mounting.</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Bioshop</td>
			<td>N/A</td>
			<td>For blocking the enzyme activity of proteinase K (PK).</td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG conjugated Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A11017</td>
			<td>For detection of primary antibody from mouse.</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG conjugated Alexa Fluor 633</td>
			<td>Invitrogen</td>
			<td>A21072</td>
			<td>For detection of primary antibody from rabbit.</td>
		</tr>
		<tr>
			<td>Intelli mixer</td>
			<td>ELMI laboratory equipment</td>
			<td>RM-2M</td>
			<td>For improving the thorughly reaction of reagents with ovaries.</td>
		</tr>
		<tr>
			<td>Laser-scanning confocal microscopy</td>
			<td>Leica Microsystems</td>
			<td>SP5</td>
			<td>For photographing of aphid embryo mounted on the slides with florecent tag.</td>
		</tr>
		<tr>
			<td>methanol&#160;</td>
			<td>Burdick and Jackson</td>
			<td>AH2304</td>
			<td>For bleaching endogenous peroxidase of embryos.</td>
		</tr>
		<tr>
			<td>Microscope slides&#160;</td>
			<td>Thermo scientific</td>
			<td>10143560</td>
			<td>For mounting of embryos. SUPERFROST ground edges, ca./env. 76 x 26 mm.</td>
		</tr>
		<tr>
			<td>Microscope Cover glasses</td>
			<td>Marienfeld</td>
			<td>0101030</td>
			<td>For mounting embryos on the slides. Size 18 x 18 mm. Thickness No. 1 (0.13 to 0.16 mm)</td>
		</tr>
		<tr>
			<td>Microscope Cover glasses</td>
			<td>Marienfeld</td>
			<td>0101050</td>
			<td>For mounting embryos on the slides. Size 22 x 22 mm. Thickness No. 1 (0.13 to 0.16 mm)</td>
		</tr>
		<tr>
			<td>mouse monoclonal anti-alphaTubulin antibody (DM1A)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-32293</td>
			<td>For labeling the distrbution of alpha-tubulin of cells.</td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For sealing the coverslips on the slides.</td>
		</tr>
		<tr>
			<td>Normal goat serum (NGS)</td>
			<td>Sigma-Aldrich</td>
			<td>G9023</td>
			<td>For blocking the non-specific binding of antibodies.</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>VWR</td>
			<td>MK262159</td>
			<td>For fixation of aphid ovaries.</td>
		</tr>
		<tr>
			<td>Phalloidin-TRITC</td>
			<td>Sigma-Aldrich</td>
			<td>P1951</td>
			<td>For labeling the distrbution of F-actin on embryos.</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>As isotonic solution for aphid ovaries.</td>
		</tr>
		<tr>
			<td>Plastic dropper</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For transfering ovaries of aphids from tube to cell tray. Size 3 ml.&#160;</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Merck</td>
			<td>124678</td>
			<td>For creating pores (punching) on the cell membrane and facilitating the access of antibodies.&#160;</td>
		</tr>
		<tr>
			<td>Pyrex spot plate</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For dissection and color reactions of aphid ovaries under microscopy. Each cavity with diameter 22 mm and&#160; depth 7 mm.</td>
		</tr>
		<tr>
			<td>SIGMAFAST 3,3&#8217;-diaminobenzidine (DAB) tablets&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D4168</td>
			<td>For developing of substrated signals. Also called DAB peroxidase substrate tablet set.</td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Leica Microsystems</td>
			<td>EZ4</td>
			<td>For dissection and observation of aphid ovaries.</td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>For creating pores (punching) on the cell membrane.&#160;</td>
		</tr>
		<tr>
			<td>VECTASHIELD Elite ABC Kit (Rabbit IgG)</td>
			<td>Vector laboratories</td>
			<td>PK-6101</td>
			<td>For enhancement of the signal. Including of biotinylated goat anti-rabbit IgG secondary antibody, A and B reagents.</td>
		</tr>
		<tr>
			<td>VECTASHIELD mounting medium</td>
			<td>Vector laboratories</td>
			<td>H1000</td>
			<td>For clearing of aphid embryos and mounting.</td>
		</tr>
	</tbody>
</table>

identification of critical conditions for immunostaining in the pea aphid embryos: increasing tissue permeability and decreasing background staining

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental studies. Like other aphids, A. pisum propagate rapidly via parthenogenetic viviparous reproduction, where the embryos develop within egg chambers in an assembly-line fashion in the ovariole. Previously we have established a robust platform of whole-mount in situ hybridization allowing detection of mRNA expression in the aphid embryos. For analyzing the expression of protein, though, established protocols for immunostaining the ovarioles of asexual viviparous aphids did not produce satisfactory results. Here we report conditions optimized for increasing tissue permeability and decreasing background staining, both of which were problems when applying established approaches. Optimizations include: (1) incubation of proteinase K (1 &#181;g/ml, 10 min), which was found essential for antibody penetration in mid- and late-stage aphid embryos; (2) replacement of normal goat serum/bovine serum albumin with a blocking reagent supplied by a Digoxigenin (DIG)-based buffer set and (3) application of methanol rather hydrogen peroxide (H2O2) for bleaching endogenous peroxidase; which significantly reduced the background staining in the aphid tissues. These critical conditions optimized for immunostaining will allow effective detection of gene products in the embryos of A. pisum and other aphids.

In this study, we performed whole-mount immunostaining on embryos of asexual pea aphids (Figure 1A). These females produce offspring parthenogenetically and viviparously. These female embryos develop within egg chambers of the ovarian tubules (ovarioles) (Figure 1B and Figure 2A). Before microscopy, the dissected ovarioles are the staining targets; however, separation of egg chambers is required for observation of embryos under a microsco...

Watch this Scientific Journal Video about Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining at JoVE.c

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

A protocol of whole-mount immunostaining on aphid embryos is presented, in which critical conditions for decreasing background staining and increasing tissue permeability are addressed. These conditions are specifically developed for effective detection of protein expression in the embryonic tissues of aphids.

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental ...

The overall goal of this immunostaining protocol is to detect protein expression in the aphid embryos. Critical conditions are identified that increase tissue permeability and decrease background staining, both of which are long-standing problems when staining embryonic tissues of aphids. The main advantage of this technique is that it provides specific conditions for effective penetration of antibodies and reduction of background staining, which usually are not described in a standard immunostaining protocol.We first had this idea of this measure when we were studying how germ cells are specified in a pea aphid a variety of insect model, for genomics and environmental studies. The laboratory strain of the pea aphid, a. pisum, was originally collected in central Taiwan, and has been reared on host plants for more than 300 generations.To germinate, soak the seeds of the host plants in tap water for three to five days at room temperature. Refill the fresh water once per day. Grow ten germinating seeds in a small pot with soil in the growth chamber under a photo period of 16 hours light, eight hours dark, at 20 degrees Celsius.About 10 days after growth begins, the height of the plants is usually more than eight centimeters. Keep each pot of plants within a one liter glass beaker and transfer adult aphids onto the plants using a paintbrush. Then, seal the beaker with an air-permeable cover, such as gauze mesh, to prevent aphids from escaping.Incubate the aphids in growth chamber under a photo-period of 16 hours light, eight hours dark, at 20 degrees Celsius, watering each pot of plants once every day. Fill one well of a spot plate with about 500 microliters of 4 percent paraformaldehyde. Place the plate under a stereomicroscope at low magnification, and submerge an adult aphid into the paraformaldehyde for dissection.Dissect the ovaries by holding the head and abdomen with one set of forceps, cutting open the dorsal cuticle of the abdomen and dragging the ovaries away from the abdominal cavity. Next, fix three pairs of ovaries in 1.5 milliliter tube containing one milliliter of paraformaldehyde at room temperature for 20 minutes, with mild shaking on a mixer. Discard the fixation buffer with a glass dropper, and then wash the ovaries with PBST three times for 10 minutes, with mild shaking.To treat the ovaries, first incubate them with Proteinase K for 10 minutes with mild shaking to increase the permeability of the embryonic tissues. Following incubation, discard the Proteinase K solution, and then wash the ovaries with 700 microliters of glycine three times for five minutes. Next, wash the ovaries with PBST twice for 10 minutes.Fix the ovaries again with fixation buffer for 15 minutes at room temperature with mild shaking. To suppress the engoenous peroxidase activity, serially dehydrate the ovaries with different percentages of methanol in 0.2 percent PBST with volume ratios of one to three, one to one, and three to one, and then further incubate the ovaries with 100 percent methanol for one hour at room temperature with mild shaking. Then, serially rehydrate the ovaries, as instructed in the text protocol.For three pairs of ovaries in a 1.5 milliliter tube, 200 microliters is the minimum volume for sample blocking and antibody staining. Incubate the ovaries with the 1x DIG-B blocking solution for four hours at room temperature, or overnight at four degrees Celsius, with mild shaking. Following incubation, discard the supernatant and replace with fresh 1x DIG-B blocking solution, containing the primary antibody at the appropriate dilution ratio.Stain the ovaries for four hours at room temperature, or overnight at four degrees Celsius with mild shaking. After washing the ovaries as described in the text protocol, discard the supernatant and replace with fresh 1x DIG-B blocking solution containing the secondary antibody at the appropriate dilution ratio, and stain the ovaries as before. For immunoflourescent staining, carry out staining in the dark, because the secondary antibody is light sensitive.The thickness of aphid embryos varies between stages of development. Mounting strategies are thus modified to fit early, mid, and late embryos. Transfer the ovaries, together with mounting medium, to the cell tray with a plastic dropper, and observe samples under a stereomicroscope at low magnification.Cut the calluses associated with the lateral oviduct using insect pins, and then transfer an isolated ovariole to an empty glass well with a dropper. Make up the final volume to between 50 and 100 microliters using mounting medium. Then, transfer an ovariole onto the slide with a glass dropper.Dissect egg chambers using insect pins. For embryos older than stage six of development, separation of egg chambers is suggested. For germaria and the first two egg chambers, separation is optional.Relocate a dissected egg chamber to a clean slide using a glass dropper. For embryos at stages one to 10 of development, slowly place a cover slip over the dissected germaria or egg chambers to avoid bubbles. Alternatively, for embryos at stage 11 to 18 of development, mount egg chambers on a slide with a one-sided cover slip bridge, and place another cover slip on top of the sample.For embryos older than stage 19 of development, mount the egg chambers on a slide with a double-sided cover slip bridge, and place another cover slip on top of the sample. Next, fill the space beneath the top cover slip with mounting media to avoid drying the sample. Mildly roll the top cover slip to obtain the right orientation for observation.Finally, seal around the edge of the cover slips, including the bridge cover slips, with nail polish before performing image analysis, as described in the text protocol. Shown here is a pair of ovaries dissected from a female adult. An ovariole is composed of one germarium, one to two O-sites, plus five to seven embryos, all of which are accommodated within egg chambers in an assembly line fashion.After paraformaldehyde fixation, digestion of ovaries in Proteinase K solution at a concentration of one microgram per milliliter for 10 minutes is highly recommended. Staining results show that signal intensity is significantly increased in the Poteinase K digested embryos. Incubation of embryos in the blocking solution from a DIG-based buffer set reduces more background staining than that of embryos in normal goat serum and bovine serum albumin.The endogenous activity of peroxidase can also result in high background staining. These studies reveal that submerging aphid embryos in methanol can suppress the peroxidase activity much more efficiently than treating embryos with hydrogen peroxide. The critical conditions revealed in this study can be applied for signal detection in both germ cells and somatic cells.Once mastered, this technique can be done in two days if it is performed properly. After watching this video, you should have a good understanding of how to carry out successful immunostaining on aphid embryos, including elevation of signal intensity, and reduction of background staining. While attempting this procedure, it's important to remember to manipulate every embryo gently to get a good shape of embryos.Don't forget that working with paraformaldehyde and DAPI can be extremely hazardous and precautions, such as gloves, should always be taken while performing this procedure. Following this procedure, other measures like in situ hybridization can be performed in order to answer additional questions, like whether messanger RNA is localized with its protein product in the embryonic cells. This method can help answer key questions in the field of insect environmental biology, such as how germ cells are specified, and how egg cells are determined in distinct reproductive phases in aphids.

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Research

JoVE Journal

Developmental Biology

9.9K Views.  National Taiwan University. The overall goal of this immunostaining protocol is to detect protein expression in the aphid embryos. Critical conditions are identified that increase tissue permeability and decrease background staining, both of which are long-standing problems when staining embryonic tissues of aphids. The main advantage of this technique is that it provides specific conditions for effective penetration of antibodies and reduction of background staining, which usually are not described in a standard immuno...

Video: Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining