The authors would like to thank Marika Walters for advice in developing hybridization in situ methods for soft tissues and Yury Goltsev for discussion of protocols for hybridization in situ of Anopheles gambiae embryos, which were subsequently adapted and modified to develop the protocol described here for hybridization in situ of Aedes and Culex embryos. We also acknowledge helpful recommendations given by Adam Par&eacute; and David Kosman. We thank Osvaldo Marinotti for scientific discussion and editing the protocol text.

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The authors have no disclosures.

The hybridization in situ and colorimetric staining protocol presented here for whole-mount mosquito tissues and embryos is a useful technique for the localization of transcripts within specific organs and cell types. These procedures are an improvement over our previously reported methods, both in streamlining extensive wash steps and providing additional technical details and reagent sources. 
 In our experience, colorimetric detection of hybridization signals is superior in sensitivity and clarity of hybridization signal compared to fluorescence-based detection schemes. Moreover colorimetric detection circumvents issues associated with signal discrimination in embryos, which are inherently auto-fluorescent. Limitations in detecting hybridization signals occur, when low-abundance transcripts are targeted and background staining is evident. Increasing the hybridization temperature to 65 &deg;C has been found to reduce background hybridization signals, but is not a suggested substitute for designing unique target-specific RNA probes.
 This protocol has been used to perform hybridization in situ of whole-mount salivary glands of Aedes aegypti, and ovaries and embryos of Anopheles gambiae, Anopheles stephensi, Ae. aegypti and Culex quinquefasciatus. This method also is applicable to other mosquito tissues, and presumably those of other insects. Additionally, we compiled for the first time comparative guidelines for the staging of embryonic development in three vector mosquitoes, Ae. aegypti, An. gambiae and Cx. fatigans. Observations have been reported for specific strains of these three species, under discreet rearing conditions. It is important to note that the developmental time course can vary for different strains of mosquito species and under different rearing conditions. Hybridizations in situ supplement on-going efforts to analyze the transcriptomes of mosquitoes and other arthropods, and may provide a better picture of the regulation of gene expression in these organisms.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>0.5 M EDTA</td>
 <td>Ambion</td>
 <td>AM9261</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>1M Tris-HCl </td>
 <td>Ambion</td>
 <td>AM9855G</td>
 <td>pH8.0</td>
 </tr>
 <tr>
 <td>10X PBS</td>
 <td>Ambion</td>
 <td>AM9625</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>20X SSC</td>
 <td>Ambion</td>
 <td>AM9763</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>1.5 ml microfuge tubes</td>
 <td>Ambion</td>
 <td>AM12400</td>
 <td>Less opaque than standard tubes; aids in visualizing samples</td>
 </tr>
 <tr>
 <td>Deionized formamide</td>
 <td>Ambion</td>
 <td>AM9342</td>
 <td>Storage at 4 &deg;C</td>
 </tr>
 <tr>
 <td>DEPC water</td>
 <td>Ambion</td>
 <td>AM9932</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Proteinase K</td>
 <td>Ambion</td>
 <td>AM2546</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>5.25% Sodium hypochlorite</td>
 <td>Austin's</td>
 <td>&nbsp; </td>
 <td>A-1 Brand</td>
 </tr>
 <tr>
 <td>T3 RNA Polymerase-Plus</td>
 <td>Ambion</td>
 <td>AM2733</td>
 <td>Storage at -20 &deg;C</td>
 </tr>
 <tr>
 <td>T7 RNA Polymerase</td>
 <td>Ambion</td>
 <td>AM2082</td>
 <td>Storage at -20 &deg;C</td>
 </tr>
 <tr>
 <td>95% Ethanol</td>
 <td>Fisher Scientific</td>
 <td>AC61511-0040</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Fisherbrand disposable polyethylene transfer pipettes</td>
 <td> Fisher Scientific</td>
 <td>13-711-7M</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>37% Formaldehyde</td>
 <td>Fisher Scientific</td>
 <td>F79-500</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>HPLC-grade methanol</td>
 <td>Fisher Scientific</td>
 <td>A452-1</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Magnesium chloride</td>
 <td>Fisher Scientific</td>
 <td>M87-500</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Microscope cover glass</td>
 <td>Fisher Scientific</td>
 <td>12-542A</td>
 <td>18 x18 mm</td>
 </tr>
 <tr>
 <td>N-Heptane</td>
 <td>Fisher Scientific</td>
 <td>H350-1</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>P-xylene</td>
 <td>Fisher Scientific</td>
 <td>O5082-500</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Pyrex 9-well Spot Plate</td>
 <td>Fisher Scientific</td>
 <td>13-748B</td>
 <td>100x85 mm</td>
 </tr>
 <tr>
 <td>Sodium chloride</td>
 <td>Fisher Scientific</td>
 <td>AC32730-0025</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Sodium hydroxide</td>
 <td>Fisher Scientific</td>
 <td>SS255-1</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Superfrost/Plus microscope slides</td>
 <td>Fisher Scientific</td>
 <td>12-550-15</td>
 <td>25x75x1.0 mm</td>
 </tr>
 <tr>
 <td>Davlyn Red Clear-liner Toupee tape</td>
 <td>Hair Direct</td>
 <td>RED-75R12</td>
 <td>Poly/Skin base material 0.75 in x 12 yd tape roll</td>
 </tr>
 <tr>
 <td>TOPOTA Cloning Kit for Sequening with One Shot Top10 chemically-competent E. coli</td>
 <td> Invitrogen</td>
 <td> K457501 K457540</td>
 <td>20 reactions 
 40 reactions</td>
 </tr>
 <tr>
 <td>Sonicated salmon sperm DNA</td>
 <td>Invitrogen</td>
 <td>15632-011</td>
 <td>Storage at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Anti-digoxigenin-AP Fab fragments</td>
 <td>Roche Applied Science</td>
 <td>1093274</td>
 <td>Storage at 4 &deg;C</td>
 </tr>
 <tr>
 <td>DIG RNA labeling mix</td>
 <td>Roche Applied Science</td>
 <td>1277073</td>
 <td>Storage at -20 &deg;C</td>
 </tr>
 <tr>
 <td>NBT/BCIP stock solution</td>
 <td>Roche Applied Science</td>
 <td>1681451</td>
 <td>Storage at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Western Blocking Reagent</td>
 <td>Roche Applied Science</td>
 <td>11921673001</td>
 <td>Storage at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Saatilene Hitech polyester mesh (330.130)</td>
 <td>Saati Print</td>
 <td>330.34 UO PW</td>
 <td>330 threads/inch, 34 micron thread diameter, orange color</td>
 </tr>
 <tr>
 <td>Glycerol</td>
 <td>Sigma</td>
 <td>G6279-1</td>
 <td>70% in PBT</td>
 </tr>
 <tr>
 <td>Heparin sodium salt</td>
 <td>Sigma</td>
 <td>H3393</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Tween 20</td>
 <td>Sigma</td>
 <td>P1379-500</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>37% Formaldehyde</td>
 <td>Ted Pella</td>
 <td>18508</td>
 <td>10 ml aliquots in amber ampoules</td>
 </tr>
 <tr>
 <td>16 oz. Solo Paper containers with lids</td>
 <td>The Paper Company</td>
 <td>SOLOKH16AJ8000</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Borosilicate glass scintillation vial with unattached screw cap</td>
 <td>VWR International</td>
 <td>66022-128</td>
 <td>20 ml case of 500 </td>
 </tr>
 <tr>
 <td>Sealed Air Bubble wrap celluar cushioning material</td>
 <td> VWR International</td>
 <td>500018-081</td>
 <td>10 foot/roll 0.188 inches thick</td>
 </tr>
 <tr>
 <td>Chicken serum</td>
<td>&nbsp; </td>
<td>&nbsp; </td>
<td>Whole blood was collected either from the wing vein or by cardiac puncture from a juvenile chicken. Blood was incubated at 37 &deg;C for 1 h until coagulated and then placed on ice for 30 min. The serum was collected and centrifuged at 3000 x g for 10 min. The resulting supernatant (clarified serum) was collected and stored at -20 &deg;C until use.</td>
 </tr>
 </tbody>
</table>
Table 4. Table of specific reagents and equipment for hybridization in situ.

hybridization in situ of salivary glands, ovaries, and embryos of vector mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Watch this Scientific Journal Video about Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes at JoVE.com

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

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Immunology and Infection

16.4K Views.  University of California, Irvine. Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.