The authors thank Weibing Tang for his great help in providing the filming lab. This article is supported by the Public Welfare Research, and special funds were received from the National Health and Family Planning of China (Grant No.201402007).

This protocol was approved by the Research Ethics Board of the Union Hospital of Huazhong University of Science and Technology.
1. Rectal Suction Biopsy
<ol>
	<li>Perform the rectal suction biopsy by a well-trained pediatric surgeon and an assistant using a Rbi2 suction rectal biopsy system after obtaining informed consent from the guardian.
	<ol>
		<li>Perform RSB on patients that have the following indications: inability to pass meconium in infants or long-term bloating with or without constipation in children; children with long-term constipation who need paraffin oil to complete defecation; intestinal obstruction or intestinal perforation with unknown reasons in children undergoing an enterostomy. 
		NOTE: The contraindications for RSB are as follows: children who have severe symptoms, who are in poor physical condition, or who cannot tolerant the RSB; children with acute-stage enterocolitis; children undergoing intestinal anastomosis without complete healing.</li>
	</ol>
	</li>
	<li>Perform a normal saline retention enema as bowel preparation 36 h before the procedure to avoid loose stools and excessive edema of the mucosa. Add magnesium sulfate solution if the child has severe constipation. Administer vitamin K (1 mg/kg) to neonates. 
	NOTE: Do not perform preoperative blood tests or administer antibiotics, as they are not required.</li>
	<li>Put patient in the lithotomy position. Coat the surface of the tube with paraffin oil. Insert the blunt-ended tube 4-7 cm into the rectum with the side hole facing the posterior or lateral walls.</li>
	<li>Apply gentle pressure on the tube to facilitate adequate adhesion of the side hole to the rectal wall. Press the trigger and withdraw the tool immediately.</li>
	<li>Use the biopsy instrument to obtain 4 suction biopsies with a diameter of 2 mm at 3 cm and 6 cm above the dental line, anteriorly and posteriorly. Use a needle to place the specimen on gauze moistened with saline.</li>
	<li>Observe the patient until 2 h after the procedure to rule out rectal bleeding. Avoid further rectal and anal manipulations in the first 24 h after RSB.</li>
</ol>
2. Preparation of the RSB Sections
<ol>
	<li>Place the rectal suction biopsies in 4% paraformaldehyde for 6 h to fix the tissue. Use a fixative volume 5 times more than the tissue volume.</li>
	<li>Dehydrate the tissue using the pathological tissue dehydrator for 11 h. The whole programmed process consists of the following 5 steps:
	<ol>
		<li>Place the tissue in formaldehyde and fix for 1 h.</li>
		<li>Place the tissue in 75% ethanol for 4 h.</li>
		<li>Place the tissue in absolute ethanol (two changes, 1 h each).</li>
		<li>Place the tissue in absolute ethanol (two changes, 30 min each).</li>
		<li>Place the tissue in xylene (three changes, 1 h each).</li>
	</ol>
	</li>
	<li>Place the tissue in paraffin wax (58-60 &#176;C) (three changes, 1 h each). Embed the RSB tissues in paraffin blocks.</li>
	<li>Trim the paraffin blocks using a microtome until the tissue is entirely exposed with a complete section plane.</li>
	<li>Cut the blocks into 0.4 &#956;m sections with a microtome.</li>
	<li>Place the sections into 45-50 &#176;C water for a few seconds each to allow the sections to spread into a flat plate.&#160;</li>
	<li>Transfer the paraffin sections onto slides.</li>
	<li>Bake the slides in a 60 &#176;C oven for 3-5 h. Avoid baking for too long, which may lead to the loss of the antigens.</li>
	<li>Place the deparaffinized slides into xylene (2 changes, 15 min each).</li>
	<li>Hydrate the slides in absolute ethanol, 95%, 80%, and 75% ethanol for 5 min each. Then, rinse them in reverse osmosis (RO)-purified water for another 5 min.</li>
</ol>
3. Antigen Retrieval
<ol>
	<li>Make working dilutions for calretinin retrieval by mixing 4 mL of EDTA antigen retrieval buffer with 200 mL of RO-purified water. Make working dilutions for S100 and PGP9.5 retrieval by mixing 1 mL of citric acid sodium citrate buffer (100x) with 99 mL of RO-purified water. 
	NOTE: Calretinin can be retrieved preferentially by EDTA retrieval solution. However, citric acid sodium citrate buffer is more suitable for the retrieval of S100 and PGP9.5.</li>
	<li>Place the slides in a coplin staining jar. Fill the jar with retrieval solution and place it into a preheated boiling water bath. After boiling for 20 min, remove the jar from the water bath and let it cool to room temperature. The cooling process takes approximately 10 min. Remove the slides and gently rinse them once with phosphate-buffered saline (PBS).</li>
	<li>Draw a circle with a marker pen around the tissue and prepare a wet box for the following procedures.</li>
</ol>
4. Blocking
<ol>
	<li>Make working dilutions by mixing 3 mL of 30% H2O2 with 27 mL of RO-purified water.</li>
	<li>Add two or three drops of 3% H2O2 solution dropwise onto the tissue to block peroxidates. Add the H2O2 solution promptly to ensure that the solution will not overflow from the circle. Perform blocking of peroxidates in a 37 &#176;C incubator for 20 min.</li>
	<li>Rinse the slides gently with PBS (three washes, 5 min each).</li>
	<li>Block the slides for 20 min at 37 &#176;C with 5% bovine serum albumin (BSA, prepared by mixing 1.5 g of BSA powder with 30 mL of RO-purified water). 
	NOTE: Blocking with 3% H2O2 can prevent 95% of nonspecific staining and blocking with 5% BSA can prevent the other 5% of nonspecific staining. Combine the two methods to obtain a reliable result.</li>
</ol>
5. Antigen-antibody Reaction
<ol>
	<li>Remove the 5% BSA and add 50 &#956;L of primary antibody (calretinin, S100, or PGP9.5) to the slide. Incubate overnight at 4 &#176;C.</li>
	<li>Wash the slide with PBS (three times, 3 min each).</li>
	<li>Add 50 &#956;L of polymer enhancer (reagent A; see Table of Materials) to the section and incubate it for 20 min in a 37 &#176;C incubator. Then, wash the slide with PBS (three times, 3 min each).</li>
	<li>Add 50 &#956;L of secondary antibody B (goat anti-rabbit/mouse IgG secondary antibody) to the section and incubate for 30 min in a 37 &#176;C incubator. Wash the section with PBS (three times, 5 min each).</li>
</ol>
6. 3,3-Diaminobenzidine and Hematoxylin Staining
<ol>
	<li>Add 850 &#956;L of RO-purified water to a 1.5 mL tube and add the staining reagents (see Table of Materials) in the order A, B, and C. Add 50 &#956;L of each reagent to obtain a total of 1 mL of 3,3-diaminobenzidine (DAB) working solution. If there are precipitates, use the solution after filtration.</li>
	<li>Add a drop of DAB working solution to the tissue section and stain for 3-10 min. Incubate the slides in DAB at room temperature until brown staining is detected. Monitor carefully using a brightfield microscope. Keep the DAB working solution away from light. Then, wash the slide with PBS for 5 min.</li>
	<li>Add a drop of hematoxylin to counterstain the nuclei for approximately 1 min. Then, hold the coplin jar and wash it under a trickle of water for approximately 30-40 s until the excess dye is removed. Be careful not to damage the tissue sections.</li>
	<li>Immerse the RSB slides in a coplin jar containing PBS for 25-30 s. Then, differentiate in 1% hydrochloric acid-ethanol for 10 s. Wash the slides with PBS for 1 min.</li>
	<li>Dehydrate the slides in the reverse order of hydration: 75%, 80%, 95%, and absolute ethanol (5 min each).</li>
	<li>Expose the slides to xylene (2 changes, 5 min each).</li>
	<li>Mount the coverslip using ultraclean mounting. Allow slides to dry before visualization using a microscope.</li>
</ol>

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N., Ekberli Agirbas, G., Tiryaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calretinin+immunohistochemistry+in+Hirschsprung's+disease:+An+adjunct+to+formalin-based+diagnosis.">Calretinin immunohistochemistry in Hirschsprung's disease: An adjunct to formalin-based diagnosis.</a> The Turkish Journal of Gastroenterology. 23 (3), 226-233 (2012).</li><li>Bachmann, L., 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=Immunohistochemical+panel+for+the+diagnosis+of+Hirschsprung&#39;s+disease+using+antibodies+to+MAP2,+calretinin,+GLUT1+and+S100.">Immunohistochemical panel for the diagnosis of Hirschsprung&#39;s disease using antibodies to MAP2, calretinin, GLUT1 and S100.</a> Histopathology. 66 (6), 824-835 (2015).</li><li>Jiang, M., 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=S100+and+protein+gene+product+9.5+immunostaining+of+rectal+suction+biopsies+in+the+diagnosis+of+Hirschsprung'+disease.">S100 and protein gene product 9.5 immunostaining of rectal suction biopsies in the diagnosis of Hirschsprung' disease.</a> American Journal of Translational Research. 8 (7), 3159 (2016).</li><li>Takawira, C., D'Agostini, S., Shenouda, S., Persad, R., Sergi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+procedures+update+on+Hirschsprung+disease.">Laboratory procedures update on Hirschsprung disease.</a> Journal of Pediatric Gastroenterology &amp; Nutrition. 60 (5), 598 (2015).</li><li>Meier-Ruge, W., 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=Acetylcholinesterase+activity+in+suction+biopsies+of+the+rectum+in+the+diagnosis+of+Hirschsprung's+disease.">Acetylcholinesterase activity in suction biopsies of the rectum in the diagnosis of Hirschsprung's disease.</a> Journal of Pediatric Surgery. 7 (1), 11-17 (1972).</li><li>Barshack, I., Fridman, E., Goldberg, I., Chowers, Y., Kopolovic, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+loss+of+calretinin+expression+indicates+aganglionosis+in+Hirschsprung's+disease.">The loss of calretinin expression indicates aganglionosis in Hirschsprung's disease.</a> Journal of Clinical Pathology. 57 (7), 712-716 (2004).</li><li>Kapur, R. 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R., Bobrow, L. G., Happerfield, L., Keeling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+PGP9.5+in+the+diagnosis+of+Hirschsprung's+disease.">Evaluation of PGP9.5 in the diagnosis of Hirschsprung's disease.</a> Journal of Pathology. 168 (1), 55 (1992).</li><li>Huang, Y., Anupama, B., Zheng, S., Xiao, X., Chen, 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+expression+of+enteric+nerve+markers+and+nerve+innervation+in+total+colonic+aganglionosis.">The expression of enteric nerve markers and nerve innervation in total colonic aganglionosis.</a> International Journal of Surgical Pathology. 19 (3), 303 (2011).</li></ol>

The authors have nothing to disclose.

Here we described a procedure using three different immunohistochemical antibodies to stain RBS sections for the diagnosis of HD. The sensitivity of our diagnostic protocol was 96.49% (95% CI, 0.88-0.99), and the specificity was 100% (95% CI, 0.97-1.00).
The most critical steps of the protocol are RSB and antigen-antibody reaction. The size of the biopsy determines the accuracy of the staining. A small biopsy will not provide enough tissue to make a precise diagnosis, while a large biopsy lead...

Hirschsprung&#39;s disease (HD) is a common congenital gut intestinal disorder characterized by a lack of ganglion cells in different segments of the distal intestinal tract1. The human enteric nervous system is formed when the invasion of the embryonic neural cells is completed. If there is a disturbance of the process and the invasion fails to complete, the distal intestine of the newborn becomes aganglionic2. This potentially fatal condition is called Hirschsprung&#39;s disease. Proliferation, motility, and gut growth are the three main components of successful colonization.
Traditional hematoxylin and eosin (H&#38;E) staining of a limited submucosal biopsy cannot attain as satisfactory of a result as H&#38;E staining of a full-thickness tissue obtained from surgery. Additionally, acetylcholinesterase (AChE) staining of rectal suction tissue is theoretically challenging due to its inadequate sensitivity, which is 91%, and complex processing of frozen sections3,4. Several other immunohistochemical markers of ganglion cells and nerve fibers that can be stained in formalin-fixed and paraffin-embedded specimens are gradually becoming the mainstream HD diagnostics. Calretinin is a vitamin D-dependent calcium-binding protein that is not expressed in the myenteric and submucosal plexus of HD-affected segments5. S100 protein is expressed in cells derived from the neural crest, such as nerve fibers and glial cells, which often present neural hypertrophy in the submucosal tissue of HD-affected segments6. Protein gene product 9.5 (PGP9.5) reliably stains nerve fibers and ganglion cells; PGP9.5 staining acts as a supplement to calretinin staining, especially in cases of isolated hypoganglionosis. Double staining with S100 and PGP9.5 can decrease the false-negative rate and increase the sensitivity. As a prerequisite, the current study aims to ensure adequate specificity and high sensitivity of this novel diagnostic method. Our novel protocol used all three markers for the discrimination of aganglionic intestine and hypertrophic nerve fibers. A prospective study of 318 children was performed by our lab and previously published without a detailed protocol7. The detailed protocol and precautions are discussed in this article. Any neonates who suffered from a severe defecation problem since birth or children with chronic constipation excluding other common diseases are potential candidates for rectal suction biopsy (RSB). Our novel protocol is suitable for staining not only RSBs but also full-thickness biopsies or surgical specimens to make a final diagnosis.

<table>
	<tbody>
		<tr>
			<td>calretinin antibody</td>
			<td>MXB Biotechnologies</td>
			<td>MAB-0716 170416405c</td>
			<td>antibody: primary antibody</td>
		</tr>
		<tr>
			<td>S-100 antibody</td>
			<td>MXB Biotechnologies</td>
			<td>Kit-0007</td>
			<td>antibody: primary antibody</td>
		</tr>
		<tr>
			<td>PGP9.5 antibody</td>
			<td>Shanghai long island antibody Co. Ltd</td>
			<td>R-0457-03</td>
			<td>antibody: primary antibody</td>
		</tr>
		<tr>
			<td>enhancer reagent</td>
			<td>MXB Biotechnologies</td>
			<td>KIT-9902-A 170416405a</td>
			<td>antibody: secondary antibody A</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit/Mouse IgG Secondary Antibody</td>
			<td>MXB Biotechnologies</td>
			<td>KIT-9902-B</td>
			<td>antibody: secondary antibody B</td>
		</tr>
		<tr>
			<td>DAB staining kit (containing reagent A B and C)</td>
			<td>MXB Biotechnologies</td>
			<td>DAB-0031</td>
			<td>staining kit</td>
		</tr>
		<tr>
			<td>Heat incubator</td>
			<td>Shanghai yiheng instrument Co. Ltd</td>
			<td>DHP-9082</td>
			<td>instrument</td>
		</tr>
		<tr>
			<td>Rbi2 suction rectal biopsy system</td>
			<td>Aus Systems Pty Ltd, South Australia, Australia</td>
			<td>CP1200 HP1000 SS1000</td>
			<td>instrument</td>
		</tr>
		<tr>
			<td>microtome</td>
			<td>Leica</td>
			<td>leica RM2016</td>
			<td>instrument</td>
		</tr>
		<tr>
			<td>citric acid sodium citrate buffer(100X)</td>
			<td>MXB Biotechnologies</td>
			<td>MVS-0101</td>
			<td>antigen retrieval buffer</td>
		</tr>
		<tr>
			<td>pathological tissue dehydrator</td>
			<td>wuhan junjie electronic Co. Ltd</td>
			<td>JT-12F</td>
			<td>instrument</td>
		</tr>
	</tbody>
</table>

diagnosis of hirschsprung's disease by immunostaining rectal suction biopsies for calretinin, s100 protein and protein gene product 9.5

Hirschsprung&#39;s disease (HD) is a congenital intestinal disease that is clinically manifested as an inability to pass meconium in infants or as long-term constipation in children. Rectal suction biopsy (RSB) to determine the absence of ganglion cells and neural hypertrophy is the most accurate test for the diagnosis of HD at present. Traditional hematoxylin-eosin staining lacks sensitivity and specificity. Acetylcholinesterase staining cannot be widely used due to its complex process. Our novel protocol of immunostaining for calretinin, S100 protein, and protein gene product 9.5 (PGP9.5), which we conducted on RSBs, exhibits high sensitivity and specificity rates of 96.49% (95% confidence interval, 0.88-0.99) and 100% (95% confidence interval, 0.97-1.00), respectively. The HD-affected segments often present as the absence of the expression of calretinin, S100 protein, and PGP9.5, which are markers of neural hypertrophy in the submucosal tissue. This protocol describes the detailed operating process of this new diagnostic method.

In total, 318 patients were enrolled in our study. All of the patients underwent RSB, and the tissues were stained for calretinin, S100, and PGP9.5. The diagnosis based on our novel protocol was HD in 97 cases, non-HD in 213 cases, and suspected HD in 8 cases. Among the 132 surgical patients, 99 patients were diagnosed with HD by immunostaining of full-thickness specimens after surgery. S100 and PGP9.5 staining showed that 92% and 93% of the 99 patients, respectively, had hypertrophied ne...

Watch this Scientific Journal Video about Diagnosis of Hirschsprung's Disease by Immunostaining Rectal Suction Biopsies for Calretinin, S100 Protein and Protein Gene Product 9.5 at JoVE.com

Diagnosis of Hirschsprung's Disease by Immunostaining Rectal Suction Biopsies for Calretinin, S100 Protein and Protein Gene Product 9.5

This protocol describes the process of immunostaining rectal suction biopsies for calretinin, S100 protein, and protein gene product 9.5. This novel adjuvant diagnostic method for Hirschsprung&#39;s disease has preferable sensitivity and specificity rates.

Hirschsprung&#39;s disease (HD) is a congenital intestinal disease that is clinically manifested as an inability to pass meconium in infants or as ...

diagnosis-hirschsprung-s-disease-immunostaining-rectal-suction

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13.2K Views.  Huazhong University of Science and Technology. This protocol describes the process of immunostaining rectal suction biopsies for calretinin, S100 protein, and protein gene product 9.5. This novel adjuvant diagnostic method for Hirschsprung's disease has preferable sensitivity and specificity rates.

Video: Diagnosis of Hirschsprung's Disease by Immunostaining Rectal Suction Biopsies for Calretinin, S100 Protein and Protein Gene Product 9.5

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult1. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3.
Amyloid &beta;-protein (A&beta;) is central to AD pathogenesis. Soluble, oligomeric A&beta; assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5. Various forms of soluble A&beta; assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6. Specific molecular recognition tools may unravel the relationships amongst A&beta; assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).
Despite emergence of aptamers as tools in modern biotechnology and medicine11, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-&beta;17, a soluble, oligomeric, &beta;-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18. Aptamers generated using monomeric and several forms of fibrillar &beta;2-microglobulin (&beta;2m) were found to bind fibrils of certain other amyloidogenic proteins besides &beta;2m fibrils19. Ylera et al. described RNA aptamers selected against immobilized monomeric A&beta;4020. Unexpectedly, these aptamers bound fibrillar A&beta;40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric A&beta;4021 generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23. Similar to previous findings17,19,20, these aptamers reacted with fibrils of A&beta; and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21. Here, we present the SELEX methodology used in production of these aptamers21.

Selection of Aptamers for Amyloid &#946;-Protein, the Causative Agent of Alzheimer's Disease

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid &beta;-protein, the causative agent of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...

Bioluminescence Imaging of Neuroinflammation in Transgenic Mice After Peripheral Inoculation of Alpha-Synuclein Fibrils

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein prionoids, which cause neuropathology within the central nervous system (CNS). Here we describe that intraglossal or intraperitoneal injection of alpha-synuclein fibrils into bigenic Tg(M83+/-:Gfap-luc+/-) mice, which overexpress human alpha-synuclein with the A53T mutation from the prion protein promoter and firefly luciferase from the promoter for glial fibrillary acidic protein (Gfap), is sufficient to induce neuropathologic disease. In comparison to homozygous Tg(M83+/+) mice that develop severe neurologic symptoms beginning at an age of 8 months, heterozygous Tg(M83+/-:Gfap-luc+/-) animals remain free of spontaneous disease until they reach an age of 22 months. Interestingly, injection of alpha-synuclein fibrils via the intraperitoneal route induced neurologic disease with paralysis in four of five Tg(M83+/-:Gfap-luc+/-) mice with a median incubation time of 229 &#177;17 days. Diseased animals showed severe deposits of phosphorylated alpha-synuclein in their brains and spinal cords. Accumulations of alpha-synuclein were sarkosyl-insoluble and colocalized with ubiquitin and p62, and were accompanied by an inflammatory response resulting in astrocytic gliosis and microgliosis. Surprisingly, inoculation of alpha-synuclein fibrils into the tongue was less effective in causing disease with only one of five injected animals showing alpha-synuclein pathology after 285 days. Our findings show that inoculation via the intraglossal route and more so via the intraperitoneal route is suitable to induce neurologic illness with relevant hallmarks of synucleinopathies in Tg(M83+/-:Gfap-luc+/-) mice. This provides a new model for studying prion-like pathogenesis induced by alpha-synuclein prionoids in greater detail.

Peripheral injection of alpha-synuclein fibrils into the peritoneum or tongue of Tg(M83+/-:Gfap-luc+/-) mice, which express human alpha-synuclein with the familial A53T mutation and firefly luciferase, can induce neuropathology, including neuroinflammation, in their central nervous system.

To study the prion-like behavior of misfolded alpha-synuclein, mouse models are needed that allow fast and simple transmission of alpha-synuclein ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...