Name: identification of critical conditions for immunostaining in the pea aphid embryos: increasing tissue permeability and decreasing background staining
Uploaded: 2016-02-02T15:00:00
Description: 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

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.

identification-critical-conditions-for-immunostaining-pea-aphid

Research

JoVE Journal

Developmental Biology

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient specificity and characteristic stemness. However, the pluripotency of iPSCs, which is essential to their functionality, must be confirmed before these cells can be used in such applications. While a rigorous characterization of iPSCs, through different cellular and functional assays is necessary to establish their pluripotency, routine assessment of pluripotency maintenance can be achieved more simply and effectively through immunocytochemical techniques. Here, we present a systematic protocol for culturing hiPSCs, in a scaled-down manner, to particularly facilitate the verification of their pluripotent state using immunocytochemistry. More specifically, this methodology encompasses an efficient and cost-effective means of growing iPSCs in serum-free conditions and plating them on small chamber slides or glass coverslips ideal for immunocytochemistry.

Regular characterization of induced pluripotent stem cells (iPSCs), to ascertain maintenance of their pluripotent state, is an important step before these cells are used for other applications. Here we describe a method for the small-scale propagation of human iPSCs specifically designed to enable their easy and routine characterization via immunocytochemistry.

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response to&#160;growth and reproduction. In insects, the larval prothoracic gland (PG) and the adult female ovary play essential roles in biosynthesizing the principal steroid hormones called ecdysteroids. These ecdysteroidogenic organs are innervated from the nervous system, through&#160;which the timing of biosynthesis is affected by environmental cues. Here we describe a protocol for visualizing ecdysteroidogenic organs and their interactive organs in larvae and adults of the fruit fly Drosophila melanogaster, which provides a suitable model system for studying steroid hormone biosynthesis and its regulatory mechanism. Skillful dissection allows us to maintain the positions of ecdysteroidogenic organs and their interactive organs including the brain, the ventral nerve cord, and other tissues. Immunostaining with antibodies against ecdysteroidogenic enzymes, along with transgenic fluorescence proteins driven by tissue-specific promoters, are available to label ecdysteroidogenic cells. Moreover, the innervations of the ecdysteroidogenic organs can also be labeled by specific antibodies or a collection of GAL4 drivers in various types of neurons. Therefore, the ecdysteroidogenic organs and their neuronal connections can be&#160;visualized simultaneously&#160;by immunostaining and transgenic techniques. Finally, we describe how to visualize germline stem cells, whose proliferation and maintenance are controlled by ecdysteroids. This method contributes to comprehensive understanding of steroid hormone biosynthesis and its neuronal regulatory mechanism.

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

We describe a protocol for dissection, fixation, and immunostaining of steroidogenic organs in Drosophila larvae and adult females to study steroid hormone biosynthesis and its regulatory mechanism. In addition to steroidogenic organs, we visualize the innervation of steroidogenic organs as well as steroidogenic target cells such as germline stem cells.

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and simple protocol to detect protein expression during craniofacial morphogenesis/pathogenesis using mouse craniofacial tissues as examples. The protocol consists of preparation and cryosectioning of tissues, indirect immunofluorescence, image acquisition, and quantification. In addition, a method for preparation and cryosectioning of undecalcified hard tissues for immunostaining is described, using craniofacial tissues and long bones as examples. Those methods are key to determine the protein expression and morphological/anatomical changes in various tissues during craniofacial morphogenesis/pathogenesis. They are also applicable to other tissues with appropriate modifications. Knowledge of the histology and high quality of sections are critical to draw scientific conclusions from experimental outcomes. Potential limitations of this methodology include but are not limited to specificity of antibodies and difficulties of quantification, which are also discussed here.

Here, we present a detailed protocol to detect and quantify protein levels during craniofacial morphogenesis/pathogenesis by immunostaining using mouse craniofacial tissues as examples. In addition, we describe a method for preparation and cryosectioning of undecalcified hard tissues from young mice for immunostaining.

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and ...