Name: A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo (Video) | JoVE Journal
Uploaded: 2022-05-06T15:00:00.000+00:00
Duration: 14 min 34 s
Description: Learn immunohistochemistry and RNA in-situ hybridization techniques for studying protein and RNA localization in Drosophila melanogaster embryos. This protocol enhances embryo attachment to slides for developmental biology research.

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5 minutes

This work was supported by the National Natural Science Foundation of China (#31771377/ 31571273/31371256), the Foreign Distinguished Scientist Program from the National Department of Education (#MS2014SXSF038), the National Department of Education Central Universities Research Fund (#GK201301001/201701005/GERP-17-45), and XZ is supported by Outstanding Doctoral Thesis Fund (#2019TS082 /2019TS079), Key Program of Shaanxi Provincial Education Department (#20JS138), the Natural Science Basic Research Program Youth Project of Shaanxi Provincial Science and Technology Department (#2020JQ-885).

1. Preparation of grape juice agar plates
<ol>
	<li>Add 5 g of agar and 5 g of sucrose to 150 mL of distilled water. Boil it using a microwave until the agar and sucrose are fully dissolved. Mix 50 mL of 100% grape juice and the solution together.</li>
	<li>Add 1 mL of 100% propionic acid to make the final concentration to 0.5% propionic acid. Pour 25 mL of the prepared solution into each plate. After the agar has solidified, store the media in 4 &#176;C.</li>
</ol>
2. Coating slides
<ol>
	<li>Pretreat slides with detergent. Wash the slides 3 times with tap water and once with distilled water. Then dry the slides in a 45 &#176;C oven for 2 h.</li>
	<li>Immerse the slides in about 450 mL of potassium dichromate sulfuric acid solution (20 g of potassium dichromate, 40 mL of distilled water, and 400 mL of concentrated sulfuric acid) for 24 hours. Wash the slides 3 times with tap water and once with distilled water.
	<ol>
		<li>Dry the slides in a 45 &#176;C oven for two hours. Immerse in 95% ethanol for more than 2 h.</li>
	</ol>
	</li>
	<li>Add 0.5 g of chromium potassium sulfate and 5 g of gelatin to 1 L of distilled water. Dissolve in a 60 &#176;C water bath before use. The chrome alum gelatin can be stored at 4 &#176;C for long periods.</li>
	<li>Drop 10 &#181;L of the chrome alum gelatin on the slide and fully and evenly cover the entire surface of the slide with the solution. Place the slide with the gelatin side up in an oven at 42 &#176;C for 48 h. The prepared slides can be stored at 4 &#176;C for later use. 
	&#8203;NOTE: This is a critical step. The complete coating is necessary. For fruit fly and its embryo, this slide coating approach appears more efficient than poly-lysine coating.</li>
</ol>
3. Drosophila embryo embedding
<ol>
	<li>Drosophila embryo collection 
	NOTE: We collected Drosophila embryos in four periods of 0-2 hours (syncytial blastoderm), 2-3 hours (cellular blastoderm), 3-12 hours (early gastrula) and 12-24 hours (late gastrula)18. The proportion of the main embryo types in each period is shown in Table 1.
	<ol>
		<li>Place 400-500 flies&#160;in a plastic collection bottle and cover the bottle with the grape juice agar plate containing yeast extract. Drosophila embryos are laid on the grape juice agar at room temperature.</li>
		<li>Collect four periods of embryos, gently adding 2 mL of PBS (137 mM NaCl, 2.7 mM KCL, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) to each plate to float the embryos. Transfer about 200 embryos to a 50 mL centrifuge tube and keep it on ice for 2 min to settle down the embryos.</li>
	</ol>
	</li>
	<li>Pre-embedding
	<ol>
		<li>Transfer 200 Drosophila embryos to a 1.5 mL centrifuge tube. Keep the tube on ice for 2 min to settle down the embryos, and then discard the supernatant with pipette carefully. Add 1 mL of 50% bleach in the tube and shake it gently for 2 min. 
		NOTE: The bleach should prepare fresh, and the amount of the bleach could vary depending on the amount of embryos collected.</li>
		<li>Gently collect embryos by pour on filter paper and wash them 3 times with 1 mL of PBS at each time.</li>
		<li>Transfer the embryos with filter paper to about 5 mL of n-heptane.</li>
		<li>Transfer the mixture to a fresh 1.5 mL centrifuge tube. Add 1 mL of methanol and shake it gently. Collect embryos after sedimentation.</li>
		<li>Remove supernatant and wash the embryos in 1 mL of PBS 3-5 times.</li>
		<li>Fix the embryos with 400 &#181;L of Bouin&#39;s solution (71% saturated picric acid, 24% formalin, 5% acetic acid) for 30 min. Wash the fixed embryos 3 times in 1 mL of PBS for 5 min each time.</li>
		<li>To aggregate embryos, transfer the samples into a rubber stopper and remove the PBS.</li>
		<li>Prepare 1.2% agarose-PBS solution and keep solution at 60 &#176;C. Add 200 &#181;L of 1.2% agarose-PBS solution onto embryos to fix them into an agar block.</li>
		<li>Take the block out from stopper and store the block in PBS. 
		NOTE: During pre-embedding approach, pro-warming is critical for successful. We strongly suggest that the centrifuge tubes and micropipette tips, which will be used in this step should be completely pre-warm before operating.</li>
	</ol>
	</li>
	<li>Paraffin embedding
	<ol>
		<li>Immerse the pre-embedded agar block in 35% ethanol, 50% ethanol, 75% ethanol, and 85% ethanol for 15 min each. Then dip 2 times in 95% ethanol and 100% ethanol. 
		NOTES: About 5-10x of block volume of each followed by a graded fixative for 15-30 minutes is necessary depending on number of embryos in the block.</li>
		<li>Immerse the embryos block in 100% ethanol and xylene (1:1 ratio) solution for 15 min.</li>
		<li>Transfer the block to xylene for 1 min to make the embryo tissue transparent.</li>
		<li>Submerge the block in xylene and paraffin (1:1 ratio) for 30 min.</li>
		<li>Submerge the block in paraffin I, paraffin II, and paraffin III for 2 h each time.</li>
		<li>Fill the embedding box with paraffin beforehand, and then gently and horizontally transfer the block into the bottom of embedding box. After the paraffin solidify naturally, the solidified paraffin block can be stored at 4 &#176;C. 
		&#8203;NOTES: The pre-embedded sample can be used in cryosection by following the following steps. After the embryos are pre-embedded with agarose as described above, the samples are simply embedded in OCT Compound. Afterwards, cryosection can be carried out according to routine cryosection protocol. Subsequently, regular staining approaches can be used on the sections.</li>
	</ol>
	</li>
</ol>
4. Hematoxylin-eosin staining
<ol>
	<li>Prepare Drosophila embryo sections. Place prepared sections in an oven at 60 &#176;C for 15 min and then submerge the sections in xylene 2 times for 10 min each. Submerge the sections once in 100% ethanol and xylene (1:1 ratio) for 2 min.</li>
	<li>Hydrate the sections in 100% ethanol I, 100% ethanol II, 95% ethanol, 85% ethanol, 75% ethanol, 50% ethanol, and in distilled water for 3 min.</li>
	<li>Stain the sections with hematoxylin solution for 2 min. Dip the slides in 1% acid alcohol solution (1 mL of hydrochloric acid, 100 mL of 70% ethanol) quickly and wash the slides in distilled water.</li>
	<li>Dip the slides in 50% ethanol, 75% ethanol, 85% ethanol, and 95% ethanol sequentially.</li>
	<li>Stain the sections with eosin solution for 1 min.</li>
	<li>Dehydrate the slides in 95% ethanol I, 95% ethanol II, 95% ethanol III, 100% ethanol I, 100% ethanol II and 100% ethanol III for 1 min each time.</li>
	<li>Clear the slides in 100% xylene I, 100% xylene II, and 100% xylene III for 5 min each time.</li>
	<li>Mount the slides with neutral balsam according to manufacturer&#39;s instructions.</li>
</ol>
5. Periodic acid-silver methenamine staining
<ol>
	<li>Deparaffinize and hydrate paraffin sections of Drosophila embryo to distilled water.</li>
	<li>Oxidize the sections in 1% periodic acid for 15 min at room temperature.</li>
	<li>Rinse the slides in distilled water 3 times for 1 min each.</li>
	<li>Incubate the slides in about 40 mL of freshly filtered silver methenamine working solution (3% methenamine, 5% silver nitrate, 5% sodium borate solution) for 1 h and 20 minutes at 60 &#176;C. Then rinse the slides in distilled water for 1 min.</li>
	<li>Tone the slides in 0.2% gold chloride for 2 min and then rinse the slides in distilled water for 1 min.</li>
	<li>Place the slides in 3% sodium thiosulfate for 3 min and then rinse the slides in distilled water for 1 min.</li>
	<li>Counterstain the slides in hematoxylin for 30 s. Wash the slides in running tap water for 3-5 min.</li>
	<li>Dehydrate the slides in 70% ethanol, 80% ethanol, 95% ethanol I, 95% ethanol II, 95% ethanol III, 100% ethanol I, and 100% ethanol II for 5 min each.</li>
	<li>Clear the slides in 100% xylene I, 100% xylene II and 100% xylene III for 5 min each.</li>
	<li>Mount the slides in neutral balsam according to manufacturer&#39;s instructions.</li>
</ol>
6. Immunohistochemistry
<ol>
	<li>Deparaffinize and rehydrate paraffin sections of Drosophila embryo to PBS following the standard protocol19.</li>
	<li>Treat slides with 0.3% H2O2 in methanol for 10 min at room temperature and avoid light.</li>
	<li>Pre-heat the appropriate antigen retrieval buffer to approximate 100 &#176;C in a container with the slides in the vegetable steamer. The container should also have a lid. Put the slides in the container. Close the lid of the steamer.
	<ol>
		<li>Keep the container in the steamer for 20 minutes from this point. Note that the lid of the slide container should not be entirely tightened so water vapor can enter during the steaming process.</li>
	</ol>
	</li>
	<li>Allow the slides return to room temperature before gently rinsing it in PBS-T (PBS with Tween-20, pH 7.2) 3 times for 5 min each, and then rinse the slides in PBS 3 times for 1 min each.</li>
	<li>Block the slides with 1% bovine serum albumin (BSA) in PBS for 60 min at room temperature.</li>
	<li>Incubate the slides with primary antibody (anti-FKBP12 at 1:1000) overnight at 4 &#176;C or at room temperature for 4 h.</li>
	<li>Wash the slides in PBS-T 4 times for 5 min each.</li>
	<li>Apply the slides with HRP labeled polymer conjugated with secondary antibodies (anti-Rabbit, 1:5,000) for 90 min at room temperature. Do this in the dark to avoid photo bleaching.</li>
	<li>Wash the slides in PBS-T 3 times for 5 min each.</li>
	<li>Incubate the slides with 5% 3,3&#39;-diaminobenzidine-tetrahydrochloride (DAB) for 2-10 min until the color of reaction products is appropriate under microscope.</li>
	<li>Counterstain the slides in hematoxylin for 30 s and wash the slides in distilled water 2 times for 5 min each.</li>
	<li>Dehydrate the slides in 50% ethanol, 70% ethanol, 90% ethanol, 95% ethanol, and 100% ethanol for 1 min each.</li>
	<li>Clear the slides in 100% xylene I, 100% xylene II and 100% xylene III for 5 min each.</li>
	<li>Mount the slides in neutral balsam according to manufacturer&#39;s instructions.</li>
</ol>
7. In-situ hybridization
NOTE: Drosophila embryo sections were prepared with 0.01% diethyl pyrocarbonate (DEPC) treated water. All accessories used for in situ hybridization apply DEPC overnight treatment in advance.
<ol>
	<li>Riboprobe preparation
	<ol>
		<li>Linearize template DNA by cutting at a restriction enzyme site downstream from the cloned insert using a restriction enzyme that creates 5&#39; overhangs. After linearization, purify the template DNA via phenol/chloroform extraction, and subsequent ethanol precipitation. Use standard protocols.</li>
		<li>Add 1 &#181;g of purified template DNA to a RNase-free centrifuge tube. Place the centrifuge tube on ice. Add enough DEPC-treated water to the total volume 13 &#181;L.
		<ol>
			<li>Then add the following reagents in order: 2 &#181;L of 10x NTP labeling mixture, 2 &#181;L of 10x transcription buffer, 1 &#181;L of protector RNase inhibitor, 2 &#181;L of RNA Polymerase SP6 or RNA Polymerase T7.</li>
		</ol>
		</li>
		<li>Mix the reaction by pipetting and centrifuge shortly. Incubate for 2 h at 37 &#176;C. 
		NOTE: Longer incubations do not increase the yield of labeled RNA. A short spin (~800 rpm) applied on the reaction tube is critical necessary before continuing to the next step. This spin will allow all contents, which could generate from the incubation, to come to the bottom of the tube.</li>
		<li>Add 2 &#181;L of RNase-free DNase I to remove template DNA and incubate for 15 min at 37 &#176;C.</li>
		<li>Add 2 &#181;L of 200 mM EDTA (pH 8.0) to stop the reaction. 
		NOTE: DIG-labeled RNA probes can be stored at -15 to -25 &#176;C in ethanol for one year.</li>
	</ol>
	</li>
	<li>Pre-hybridization of Drosophila embryo section
	<ol>
		<li>Place sections of Drosophila embryo in a 42 &#176;C oven for 48 h.</li>
		<li>Deparaffinize the slides in 100% xylene for 10 min twice.</li>
		<li>Hydrate the slides by sequentially submerging in 100% ethanol, 95% ethanol, 90% ethanol, 70% ethanol, and 50% ethanol for 5 min.</li>
		<li>Incubate the slides in PBS for 5 min 3 times to remove the fixative solution/Bouin&#39;s solution from the tissue.</li>
		<li>Wash the slides with 100 mmol/L glycine/PBS solution 2 times for 5 min each.</li>
		<li>Wash the slides with PBS 2 times for 5 min each.</li>
		<li>Incubate the slides with 0.3% Triton X-100 in PBS for 15 min to permeabilize the membranes.</li>
		<li>Wash the slides with PBS 3 times for 5 min each.</li>
		<li>Incubate the slides in 15 &#181;g/mL RNase-free proteinase K for 30 minutes at 37 &#176;C.</li>
		<li>Incubate the slides with 4% paraformaldehyde in PBS for 3 min to stop the digestion.</li>
		<li>Wash the slides with PBS 2 times for 5 min each.</li>
		<li>Incubate the slides in 100 mM triethanolamine buffer (pH 8.0) with 0.25% (v/v) acetic anhydride 2 times for 5 min each.</li>
		<li>Add DEPC water into the slide moisture chamber. Add 20 &#181;L of pre-hybridization solution (containing 100 &#181;g/mL of salmon sperm DNA) on the slides. Put the slides into the slide moisture chamber. Incubate the slides at 42 &#176;C for 4 h. 
		NOTE: It is necessary to ensure the tightness of the lid of the wet box and that the wet box is always kept in a horizontal to prevent volatilization and deviation from the tissue of the pre-hybridization solution and the followed hybridization solution.</li>
	</ol>
	</li>
	<li>Hybridization of Drosophila embryo section
	<ol>
		<li>Remove the pre-hybridization solution and wash the slides with 0.2x SSC (150 mM sodium chloride, 15 mM sodium citrate, pH 7.0) 3 times for 5 min each. Wipe off excess 0.2x SSC around the tissue samples.</li>
		<li>Apply 30 &#181;L of probe hybridization solution (containing 2-6 ng of digoxin-labeled RNA probe by diluting in pre-hybridization solution) with a pipette. Incubate the slides in the slide moisture chamber at 42 &#176;C for approximately 20 h. 
		NOTE: Be sure on the cap-tightness as the suggestion of the last note. Otherwise, the dry-up caused from the leakage of either pre-hybridization or pre-hybridization will generate dirty background including pseudo-positive in slides.</li>
	</ol>
	</li>
	<li>Hybridization
	<ol>
		<li>Remove the hybridization solution and wash the slides with 4x SSC solution 4 times for 15 min each at 37 &#176;C.</li>
		<li>Wash the slides in 2x SSC solution with 20 &#181;g/mL of RNase A for 30 min at 37 &#176;C.</li>
		<li>Wash the slides in 1x SSC solution for 15 min at 37 &#176;C.</li>
		<li>Wash the slides in 0.5x SSC solution for 15 min at 37 &#176;C.</li>
		<li>Wash the slides in 0.1x SSC solution for 15 min at 37 &#176;C.</li>
		<li>Wash the slides with PBS 3 times for 5 min each.</li>
		<li>Wash the slides in washing buffer (100 mM maleic acid, 150 mM NaCl, 0.3% (v/v) Tween 20, pH 7.5) for 1 min.</li>
		<li>Incubate the slides in 100 mL of freshly prepared blocking solution (2% sheep serum in maleic acid buffer without Tween 20) for 30 minutes.</li>
		<li>Incubate the slides in 20 mL of antibody solution for 45 minutes.
		<ol>
			<li>Centrifuge the antibody for 5 min at 10,000 rpm in the original vial prior to each use, and pipet the necessary amount carefully from the surface. Dilute anti-digoxigenin-AP 1:5000 (150 mU/mL) in blocking solution.</li>
		</ol>
		</li>
		<li>Wash the slides in 100 mL of washing buffer 2 times for 15 minutes each to remove unbound antibody conjugate.</li>
		<li>Equilibrate the slides in 20 mL of detection buffer (100 mM Tris-HCl, 100 mM NaCl, pH 9.5) for 2-5 min.</li>
		<li>Incubate the slides with 10 mL of freshly prepared color substrate solution in the slide moisture chamber. The reaction begins within a few minutes and is complete after 16 h. Do not shake or mix while color is developing.</li>
		<li>Wash the slides with 50 mL of distilled water for 5 min to stop the reaction.</li>
		<li>Dry the sample overnight in the dark.</li>
		<li>Dehydrate the slides in 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, 100% ethanol I and 100% ethanol II for 3 min each.</li>
		<li>Clear the slides in 100% xylene I, 100% xylene II and 100% xylene III for 20 min each.</li>
		<li>Mount the slides with neutral balsam.</li>
		<li>Document images and analyze with the inverted microscopy and manufacturer software. Perform distribution analysis using ImageJ according to density of positive signal either along longitudinal axis or within differential zone of embryos. 
		NOTE: During the incubation with the freshly prepared color substrate solution in the step 7.4.12, the reaction color can be seen even with a bare eye. This observation on how color developing can be inspected under stereoscope. Once reaction color is reasonable and the step 7.4.13 can be carried out to stop the reaction.</li>
	</ol>
	</li>
</ol>

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<li>Wehrens, X. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FKBP12.6+deficiency+and+defective+calcium+release+channel+%28ryanodine+receptor%29+function+linked+to+exercise-induced+sudden+cardiac+death%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death.</a> Cell. 113 (7), 829-840 (2003).</li>
<li>Wehrens, X. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protection+from+cardiac+arrhythmia+through+ryanodine+receptor-stabilizing+protein+calstabin2%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Protection from cardiac arrhythmia through ryanodine receptor-stabilizing protein calstabin2.</a> Science. 304 (5668), 292-296 (2004).</li>
<li>Bellinger, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Remodeling+of+ryanodine+receptor+complex+causes+%26%2334%3Bleaky%26%2334%3B+channels%3A+a+molecular+mechanism+for+decreased+exercise+capacity%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Remodeling of ryanodine receptor complex causes &#34;leaky&#34; channels: a molecular mechanism for decreased exercise capacity.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (6), 2198-2202 (2008).</li>
<li>Maruyama, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FKBP12+is+a+critical+regulator+of+the+heart+rhythm+and+the+cardiac+voltage-gated+sodium+current+in+mice%5BTitle%5D)+AND+%22Circulation+Research%22%5BJournal%5D)">FKBP12 is a critical regulator of the heart rhythm and the cardiac voltage-gated sodium current in mice.</a> Circulation Research. 108 (9), 1042-1052 (2011).</li>
<li>Xu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FKBP12+is+the+only+FK506+binding+protein+mediating+T-cell+inhibition+by+the+immunosuppressant+FK506%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">FKBP12 is the only FK506 binding protein mediating T-cell inhibition by the immunosuppressant FK506.</a> Transplantation. 73 (11), 1835-1838 (2002).</li>
<li>Zalk, R., Marks, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ca2%2B+release+channels+join+the+%27resolution+revolution%27%5BTitle%5D)+AND+%22Trends+in+Biochemical+Sciences%22%5BJournal%5D)">Ca2+ release channels join the 'resolution revolution'.</a> Trends in Biochemical Sciences. 42 (7), 543-555 (2017).</li>
</ol>

The authors have nothing to disclose.

RyRs and IP3Rs mediated calcium signaling is a fundamental pathway in many physiological and pathological process of both vertebrate and invertebrate animals1,2,3,4. In humans, point mutations, such as CPVT-associated R4496C mutation, in the RyR2 gene lead to calcium leakage from the sarcoplasmic reticulum of cardiomyocyte, resulting in cardiac dysfunction. These mutations present as arrhythmia...

Calcium induced calcium release signaling (CICR) plays critical role in many biological processes, such as skeletal/smooth muscle and cardiac vascular function, cell proliferation and apoptosis, development, aging, neuronal synaptic plasticity, and regeneration1,2,3,4,5,6. Ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs) are major players in the calcium signaling pathway controlled by their regulators protein kinase A (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII), FK506 binding proteins (FKBPs), calsequestrin (CSQ), triadin, and junctin1,2,3,4,5,6. Abnormal human expression and mutations in these proteins can lead to pathological physiology such as arrhythmias7 and oncogenic proliferation8,9.
FKBPs regulate the calcium release from the endoplasmic reticular (ER) by RyRs. This process is essential for the mechanism of contraction,&#160;and thus responsible for all mechanical movement generated by myosin-actin contraction through calcium-induced calcium release along with embryonic RyRs1,2. In mouse models, the lack of RyR2 and its regulator FKBP12/Calstabin is invariably lethal, either during embryonic development or in the early postnatal period10,11,12. FKBP12/Calstabin knockout mice exhibit critical cardiac defects with irregular excitation- contraction coupling (EC) and cerebral edema during embryonic development. This indicates that FKBP12/Calstabin plays an essential role in regulating RyR2 channel expression, which is important both to cardiac and cerebral development10.
RyR conducted calcium sparks were initially discovered in the zygote formation phase of fertilized Medaka eggs13,14. However, few investigations have been performed on the function of calcium signaling in early embryonic development. In Drosophila melanogaster, results obtained from DmFKBP12 S107 mutants provide strong evidence supporting the importance of this gene for larval development and a healthy life span, which is attributed to its function against oxidative stress15,16. Recently, we identified the dynamic localization of FKBP12/Calstabin protein and messenger RNA during early Drosophila melanogaster development17. Using the approaches described in this methodology, we were able to track the expression of FKBP12/Calstabin in D. melanogaster during the syncytial blastoderm (0-2 h), cellular blastoderm (2-3 h), early gastrula (3-12 h), and late gastrula (12-24 h). In this paper, we present the detailed protocols of every approach in the previous study, including pre-embryo embedding for classic paraffin sectioning, pre-coating slide treatment for embryonic sections, histo-chemistry staining and immunostaining, and mRNA in-situ hybridization for identification of gene expression.

<table><tbody><tr><td>-20&amp;deg;C Refrigerator</td><td>Meiling Biology &amp;amp;Medical</td><td>DW-YL270</td><td>Used for regent storage</td></tr><tr><td>-80&amp;deg;C Ultra low temperature refrigerator</td><td>Thermo</td><td>Forma 90 Series</td><td>Used for regent storage</td></tr><tr><td>Agar</td><td>Sigma-Aldrich</td><td>WXBB6360V</td><td>Preparation of grape juice agar plates</td></tr><tr><td>Anti-Digoxigenin-AP, Fab fragments</td><td>Roche</td><td>11093274910</td><td>For the detection of digoxigenin-labeled compound</td></tr><tr><td>Biochemical incubator</td><td>Shanghai Bluepard Instruments</td><td>LRH-250</td><td>&lt;em&gt;In-situ &lt;/em&gt;Hybridization</td></tr><tr><td>Bouin&#039;s solution</td><td>Sinopharm Chemical Reagent at Beijing</td><td>69945460</td><td>Drosophila Embryo Embedding</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>540BH07808</td><td>&lt;em&gt;In-situ&lt;/em&gt; Hybridization</td></tr><tr><td>Centrifuge tube</td><td>Denville</td><td>C-2170</td><td>Drosophila Embryo Collection</td></tr><tr><td>Chrome Alum</td><td>Sinopharm Chemical Reagent at Beijing</td><td>10001018</td><td>Coating Slides</td></tr><tr><td>Constant temperature water bath</td><td>Jintan Henfeng Instruments</td><td>KW-1000DC</td><td>Hematoxylin-Eosin Staining, Immunohistochemistry, In-situ Hybridization and Periodic Acid-Silver Methenamine Staining</td></tr><tr><td>Dako REAL EnVision Detection System</td><td>Dako</td><td>K5007</td><td>In immunohistochemical reaction or in situ hybridization reaction, it binds to the primary antigen antibody, and the target is labeled by staining.</td></tr><tr><td>DEPC</td><td>Sigma-Aldrich</td><td>D5758</td><td>In-situ Hybridization</td></tr><tr><td>DIG RNA Labeling Kit</td><td>Roche</td><td>11093274910</td><td>RNA labeling with diagoxigenin-UTP by in vitro transcription with SP6 and T7 RNA polymerase</td></tr><tr><td>&lt;em&gt;Drosophila melanogaster&lt;/em&gt;</td><td>Bloomington Stock Center</td><td>BDSC_16799, BDSC_19894, BDSC_11664</td><td>The stocks of &lt;em&gt;Drosophila melanogaster &lt;/em&gt;mutant</td></tr><tr><td>Electric blast drying oven</td><td>Tianjin Taiste Instruments</td><td>101-0AB</td><td>For coating slides and paraffin embedding</td></tr><tr><td>Eosin</td><td>Sigma-Aldrich</td><td>230251</td><td>Hematoxylin-Eosin Staining</td></tr><tr><td>Ethanol</td><td>Sinopharm Chemical Reagent at Beijing</td><td>100092680</td><td>Paraffin Embedding, Hematoxylin-Eosin Staining, Immunohistochemistry, In-situ Hybridization and Periodic Acid-Silver Methenamine Staining</td></tr><tr><td>Gelatin</td><td>Sinopharm Chemical Reagent at Beijing</td><td>10010328</td><td>Coating Slides</td></tr><tr><td>Gold chloride</td><td>Sigma-Aldrich</td><td>379948</td><td>Periodic Acid-Silver Methenamine Staining</td></tr><tr><td>Hematoxylin</td><td>Sigma-Aldrich</td><td>H3136</td><td>Hematoxylin-Eosin Staining</td></tr><tr><td>High Pure PCR Product Purification Kit</td><td>Roche</td><td>11732668001</td><td>For purification of PCR products</td></tr><tr><td>Intelligent constant temperature and humidity box</td><td>Ningbo Jiangnan Instruments</td><td>HWS</td><td>For fly maintenance</td></tr><tr><td>LE Agarose</td><td>HyAgarose</td><td>14190108029</td><td>Pre-embedding</td></tr><tr><td>Methanol</td><td>Sinopharm Chemical Reagent at Beijing</td><td>10014108</td><td>Drosophila Embryo Collection</td></tr><tr><td>Microscope</td><td>ZEISS</td><td>Observer.A1</td><td>Hematoxylin-Eosin Staining, Immunohistochemistry, In-situ Hybridization and Periodic Acid-Silver Methenamine Staining</td></tr><tr><td>Microscope Slides</td><td>MeVid Labware Manufacturing</td><td>P105-2001</td><td>Coating Slides</td></tr><tr><td>Neutral Gum</td><td>Sinopharm Chemical Reagent at Beijing</td><td>10004160</td><td>Hematoxylin-Eosin Staining</td></tr><tr><td>N-heptane</td><td>Sinopharm Chemical Reagent at Beijing</td><td>40026768</td><td>Drosophila Embryo Collection</td></tr><tr><td>Paraffin slicer</td><td>Huahai science instrument</td><td>HH-2508III</td><td>&lt;em&gt;In-situ&lt;/em&gt; Hybridization</td></tr><tr><td>Paraffin</td><td>Sinopharm Chemical Reagent at Beijing</td><td>69019461</td><td>Paraffin Embedding</td></tr><tr><td>pH/mV Meter</td><td>Sartorius</td><td>PB-10</td><td>For determing the pH value of a solution</td></tr><tr><td>Silver nitrate</td><td>Sinopharm Chemical Reagent at Beijing</td><td>10018461</td><td>Periodic Acid-Silver Methenamine Staining</td></tr><tr><td>Ultrapure water meter</td><td>Thermo</td><td>AFXI-0501-P</td><td>&lt;em&gt;In-situ&lt;/em&gt; Hybridization</td></tr><tr><td>Xylene</td><td>Sinopharm Chemical Reagent at Beijing</td><td>10023418</td><td>Paraffin Embedding</td></tr></tbody></table>

a protocol for immunohistochemistry and rna in-situ distribution within early drosophila embryo

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

The figures describe protocols used to overcome the challenge of attaching high lipid and chitin-containing chorion Drosophila embryos (Table 1) to the glass slide surface for examination and experimentation. Utilizing the chrome alum gelatin slide-coating method shown in Figure 1, we enhanced the attachment of Drosophila embryos onto the surface of slides while the embryo pre-embedding method shown in Figure 2 allows for effic...

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...

a-protocol-for-immunohistochemistry-rna-situ-distribution-within

Research

JoVE Journal

Developmental Biology

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Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

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Drosophila Pupal Abdomen Immunohistochemistry

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Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae.

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

Presented here is a protocol to microinject and simultaneously image multiple Drosophila embryos during embryonic development using a plate-based, high content imager.

Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer

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Direct Cryosectioning of Drosophila Heads for Enhanced Brain Fluorescence Staining and Immunostaining

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A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

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