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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.&#160;
1. Preparation of the working solutions and buffers
NOTE: On the morning of the assay, prepare the following solutions. This calculation is based on the necessary solutions to process two brain regions (i.e., the prefrontal cortex [PFC] and hippocampus [HPC]) from 12 mice.
<ol>
	<li>Prepare artificial cerebrospinal fluid (aCSF, pH = 7.4) as mentioned in Table 1. Dispense 750 &#181;L of aCSF into each sampling tube, and place them in the metal temperature block on ice to pre-chill the buffer.</li>
	<li>Prepare the lysis buffer as mentioned in Table 2. Store on ice (400 &#181;L to be used per sample).</li>
	<li>Prepare a 52 mM Bis(sulfosuccinimidyl)suberate or BS3 stock solution (26x) in 5 mM sodium citrate buffer (pH = 5.0).
	<ol>
		<li>First, prepare 100 mM citric acid (stock A) and 100 mM sodium citrate (stock B).</li>
		<li>Dilute stock A and stock B at a ratio of 1:20 with deionized water. Add 100 &#181;L each to 1.9 mL of water to prepare 5 mM citric acid (stock C) and 5 mM sodium citrate (stock D), respectively.</li>
		<li>Mix 410 &#181;L of stock C and 590 &#181;L of stock D to prepare a 5 mM sodium citrate buffer (pH = 5.0) (solution E, 1 mL).</li>
		<li>Confirm the pH of solution E using a pH indicator strip.</li>
		<li>Dissolve 24 mg of BS3 in 806.4 &#181;L of solution E by vortexing for 30 s to prepare the BS3 stock solution (26x).</li>
		<li>Prepare another 1 mL of solution E to use as vehicle control for the non-crosslinking samples. 
		&#160;&#8203;NOTE: Prepare BS3 stock solution when everything else is ready, and the experiment is about to begin. The BS3 should be stored desiccated at 4 &#176;C until use. Once reconstituted, BS3 remains active only for approximately &#8804;3 h. As the pH of the 5 mM sodium citrate buffer (solution E) is reported to rise over time, causing accelerated BS3 hydrolysis, it is recommended that solution E is prepared fresh from stock solutions A and B. Due to the limited solubility of BS3 at low temperatures, the reconstituted BS3 should be kept at RT. Use the reconstituted BS3 within 3 h, and do not freeze/thaw or reuse the reconstituted BS3.</li>
	</ol>
	</li>
</ol>
2. Dissection of brain tissues
NOTE: From this step on, at least two people should work together in a coordinated manner. While one person focuses on the animal dissection, the other person should work as a timekeeper and help coordinate the assay.
<ol>
	<li>Bring the first animal for dissection from the housing area to the dissection room. 
	&#160;NOTE: As acute stressors (e.g., a novel environment, the smell of blood) may affect the brain protein dynamics, the animals should be kept in their home cages placed far from the dissection area and then be brought individually into the dissection room for immediate decapitation.</li>
	<li>Euthanize the mouse by cervical dislocation followed by decapitation. Remove the brain rapidly out of the skull and submerge it in ice-cold PBS in a Petri dish for 10-15 s (Figure 1). 
	&#160;NOTE: The animals are not anesthetized for BS3 experiments since any anesthetic agent could potentially influence the surface presentation level of the neurotransmitter receptors.</li>
	<li>Place the chilled brain into the brain matrix on ice, with the ventral side of the brain facing up (Figure 2).</li>
	<li>Insert the first razor blade through the border between the olfactory bulb and the olfactory peduncle to cut the brain coronally (Figure 3). Using three to four additional razor blades, serially cut the anterior part of the brain coronally with 1 mm intervals.</li>
	<li>Lift the coronal slices off the brain matrix by holding all the inserted razor blades together, leaving the posterior part of the brain behind in the brain matrix. Use forceps to separate the razor blades from one another, and place them on the flat, chilled surface with the brain slice facing up (Figure 4).</li>
	<li>Identify the slices containing the region of interest. For sampling the PFC, choose the second and third slices posterior to the first slice containing the olfactory peduncle.</li>
	<li>Remove the region of interest using a tissue punch, put it aside on the chilled razor blade, and evenly divide the tissue into two (e.g., tissues from the left versus right hemisphere, with one half to be used for the BS3 crosslinking reaction and the other half for the no-crosslinking control if the target protein of interest is equally expressed in both hemispheres).</li>
	<li>Mince each tissue into pieces on the razor blade using the fine tip of forceps with multiple vertical motions against the blade instead of mashing or grinding the tissues. This will maximize the surface area accessible to BS3 without severely compromising the membrane integrity of the cells. Immediately after mincing, transfer the minced tissues into the appropriate tubes (see step 3.3).</li>
</ol>
3. Crosslinking reaction
<ol>
	<li>Bring the next animal for dissection from the housing area to the dissection room when the dissection of the brain of the previous mouse is about to finish (step 2.10).</li>
	<li>Spike the tube pre-chilled on ice with 30 &#181;L of BS3 solution (26x) or vehicle solution E right before the minced tissues are ready to be transferred into the appropriate tubes. Change the pipet tips in between tubes to ensure no BS3 is contaminated in the no-crosslinking control tubes.</li>
	<li>Transfer the minced tissues (from step 2.8) into the appropriate tubes, and then start dissecting the next animal (step 2.2).</li>
	<li>Invert and mix the tube to break the tissue chunks apart into smaller pieces, and start incubating the samples on the tube rotator in the cold room for 30 min to 2 hours. Record the start time of the BS3 incubation for each tube. See the discussion section for the optimal incubation time.</li>
	<li>Quench the reaction by spiking the tube with 78 &#181;L of 1 M glycine and further incubate the sample for 10 min at 4 &#176;C with constant rotation. Record the start and end time of quenching for each tube. Continue assisting the person focusing on the animal dissection by bringing the next animal (step 3.1) and helping with crosslinking (step 3.2). 
	&#160;&#8203;NOTE: Treat each sample at the exact same time across all the samples. If the dissection room is far from the cold room, it is highly recommended that a third person be recruited to participate in the sample incubation and quenching in the cold room.</li>
</ol>
Table 1: The composition of artificial cerebrospinal fluid.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Working conc.</td>
			<td>Stock solution</td>
			<td>Amount of stock solution to dispense</td>
		</tr>
		<tr>
			<td>1.2 mM CaCl2</td>
			<td>480 mM (400x)*</td>
			<td>100 &#956;L</td>
		</tr>
		<tr>
			<td>20 mM HEPES</td>
			<td>1 M (50x)</td>
			<td>800 &#956;L</td>
		</tr>
		<tr>
			<td>147 mM NaCl</td>
			<td>5 M (34x)</td>
			<td>1176.5 &#956;L</td>
		</tr>
		<tr>
			<td>2.7 mM KCl</td>
			<td>1.08 M (400x)</td>
			<td>100 &#956;L</td>
		</tr>
		<tr>
			<td>1 mM MgCl2</td>
			<td>400 mM (400x)</td>
			<td>100 &#956;L</td>
		</tr>
		<tr>
			<td>10 mM Glucose&#160;</td>
			<td>&#160;2.5 M (250x)</td>
			<td>160 &#956;L</td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td></td>
			<td>37.563 mL</td>
		</tr>
		<tr>
			<td>Total</td>
			<td></td>
			<td>40 mL</td>
		</tr>
		<tr>
			<td colspan="3">* CaCl2 stock should be freshly prepared on the day of the experiment.</td>
		</tr>
	</tbody>
</table>
Table 2: The composition of the lysis buffer
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Working conc.</td>
			<td>Stock solution</td>
			<td>Amount of stock solution to dispense</td>
		</tr>
		<tr>
			<td>25 mM HEPES</td>
			<td>1 M (40x)</td>
			<td>500 &#956;L</td>
		</tr>
		<tr>
			<td>500 mM NaCl</td>
			<td>5 M (10x)</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>2 mM EDTA</td>
			<td>0.5 M (250x)</td>
			<td>80 &#956;L</td>
		</tr>
		<tr>
			<td>1 mM DTT</td>
			<td>1 M (1000x)</td>
			<td>20 &#956;L</td>
		</tr>
		<tr>
			<td>0.1% NP-40</td>
			<td>10% (100x)</td>
			<td>200 &#956;L</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>100x</td>
			<td>200 &#956;L</td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td></td>
			<td>17 mL</td>
		</tr>
		<tr>
			<td>Total</td>
			<td></td>
			<td>20 mL</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 M EDTA, pH 8.0</td>
			<td>Invitrogen</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M HEPES</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>10x TBS</td>
			<td>Bio-Rad</td>
			<td>1706435</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5 M (45%, w/v) Glucose</td>
			<td>Sigma</td>
			<td>G8769</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>4x SDS sample buffer (Laemmli)</td>
			<td>Bio-Rad</td>
			<td>1610747</td>
			<td></td>
		</tr>
		<tr>
			<td>Bis(sulfosuccinimidyl)suberate (BS3)</td>
			<td>Pierce</td>
			<td>A39266</td>
			<td>No-Weigh Format; 10 x 2 mg</td>
		</tr>
		<tr>
			<td>Brain matrix</td>
			<td>Ted Pella</td>
			<td>15003</td>
			<td>For mouse, 30 g adult, coronal, 1 mm</td>
		</tr>
		<tr>
			<td>Calcium chloride (CaCl2)</td>
			<td>Sigma</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved probe</td>
			<td>Fine Science Tools</td>
			<td>10088-15</td>
			<td>Gross Anatomy Probe; angled 45</td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td>milli-Q</td>
			<td>EQ 7000</td>
			<td>Ultrapure water [resistivity 18.2 M&#937;&#183;cm @ 25 &#176;C; total organic carbon (TOC) &#8804; 5 ppb]</td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>10197777001</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper (3MM)</td>
			<td>Whatman</td>
			<td>3030-917</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps (large)</td>
			<td>Fine Science Tools</td>
			<td>11152-10</td>
			<td>Extra Fine Graefe Forceps</td>
		</tr>
		<tr>
			<td>Forceps (small)</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td>Dumont #5 Forceps</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma</td>
			<td>W328707</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride&#160;</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonidet-P40, substitute (NP-40)</td>
			<td>SantaCruz</td>
			<td>68412-54-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors (large)</td>
			<td>Fine Science Tools</td>
			<td>14007-14</td>
			<td>Surgical Scissors - Serrated</td>
		</tr>
		<tr>
			<td>Scissors (small)</td>
			<td>Fine Science Tools</td>
			<td>14060-09</td>
			<td>Fine Scissors - Sharp</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue punch (ID 1 mm)</td>
			<td>Ted Pella</td>
			<td>15110-10</td>
			<td>Miltex Biopsy Punch with Plunger, ID 1.0 mm, OD 1.27 mm</td>
		</tr>
		<tr>
			<td>Tube rotator (LabRoller)</td>
			<td>Labnet</td>
			<td>H5000</td>
			<td></td>
		</tr>
	</tbody>
</table>

crosslinking neuronal surface proteins in the mouse brain using a chemical crosslinking assay

Source: Sumitomo, A., et al. <a href="https://app.jove.com/v/65063">BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABAA Receptor Presentation in the Rodent Brain.</a> J. Vis. Exp. (2023)
This video demonstrates the crosslinking of neuronal surface proteins using Bis-sulfosuccinimidyl suberate or BS3. Isolated and chilled mouse brain is sectioned coronally. The desired region is extracted from the sections, minced, and treated with BS3. BS3 crosslinks surface proteins like GABA receptors, leaving internal proteins unaltered. The reaction is quenched with glycine before processing the samples for further analysis.

<img alt="Figure 1" src="/files/ftp_upload/23213/23213_Figure1.jpg" /> 
Figure 1: Whole mouse brain dissected out of the skull and placed in ice-cold PBS. Immediately after the whole brain was removed from the skull, it was submerged in ice-cold PBS for 10-15 s in a Petri dish on ice. This slows down brain metabolism and minimizes the protein trafficking and degradation while also helping the tissue to become harder, which makes the subsequent brain slicing easier.</...

Crosslinking Neuronal Surface Proteins in the Mouse Brain using a Chemical Crosslinking Assay

Source: Sumitomo, A., et al. BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABAA Receptor Presentation in the ...

Begin with an isolated mouse brain in an ice-cold buffer.
Transfer the brain to a brain matrix, with the ventral side facing up.
Insert razor blades to serially cut the anterior part of the brain.
Lift the slices by holding the blades together, leaving the posterior part behind. Separate the razor blades and place them on a chilled surface with brain slices facing up.
Select the desired slice and extract the region of interest.
Divide and mince the tissue.
Transfer the minced tissue into a tube containing cerebrospinal fluid supplemented with the chemical BS3.
Invert and mix the tube to break the tissue into smaller pieces, then incubate with rotation.
BS3, a cell membrane-impermeable chemical, crosslinks neuronal surface proteins like GABA receptors while internal GABA receptors remain unaltered.
Add glycine to quench the crosslinking reaction.
The sample is ready for evaluation of surface GABA receptor levels.

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24 visualizações. Source: Sumitomo, A., et al. BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABAA Receptor Presentation in the Rodent Brain. J. Vis. Exp. (2023) This video demonstrates the crosslinking of neuronal surface proteins using Bis-sulfosuccinimidyl suberate or BS3. Isolated and chilled mouse brain is sectioned coronally. The desired region is extracted from the sections, minced, and treated with BS3. BS3 crosslinks surface proteins like GABA receptors, leavin...

Vídeo: Crosslinking Neuronal Surface Proteins in the Mouse Brain using a Chemical Crosslinking Assay - Conceito

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility1-7. To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent &#945;-bungarotoxin with cells expressing constructs containing an &#945;-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the &#945; subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity8,9. Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors2,10. In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP11) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)12. The BBS is 3&#39; of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.

Here we demonstrate the use of fluorescent Alexa dye coupled to &#945;-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds &#945;-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.

Usando um α-Bungarotoxina Binding Tag Site para Estudar GABA um receptor de membrana Localização e Tráfico

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

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Anti-RDL and Anti-mGlutR1 Receptors Antibody Testing in Honeybee Brain Sections using CRISPR-Cas9

Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a gene editing technique widely used in studies of gene function. We use this method in this study to check for the specificity of antibodies developed against the insect GABAA receptor subunit Resistance to Dieldrin (RDL) and a metabotropic glutamate receptor mGlutR1 (mGluRA). The antibodies were generated in rabbits against the conjugated peptides specific to fruit flies (Drosophila melanogaster) as well to honeybees (Apis mellifera). We used these antibodies in honeybee brain sections to study the distribution of the receptors in honeybee brains. The antibodies were affinity purified against the peptide and tested with immunoblotting and the classical method of preadsorption with peptide conjugates to show that the antibodies are specific to the corresponding peptide conjugates against which they were raised. Here we developed the CRISPR-Cas9 technique to test for the reduction of protein targets in the brain 48 h after CRISPR-Cas9 injection with guide RNAs designed for the corresponding receptor. The CRISPR-Cas9 method can also be used in behavioral analyses in the adult bees when one or multiple genes need to be modified.

Presented here is a protocol to use the CRISPR-Cas9 system for reducing the production of a protein in the adult honeybee brain to test antibody specificity.

Testes anticorpos anti-RDL e Anti-mglutr1 em seções cerebrais de abelhas usando CRISPR-Cas9

Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a gene editing technique widely used in studies of ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

Utilizando Receptores pHluorin-tag para monitorar subcelular Localização e Tráfico

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

Crosslinking Neuronal Surface Proteins in the Mouse Brain using a Chemical Crosslinking Assay

Transcrição

Crosslinking Neuronal Surface Proteins in the Mouse Brain using a Chemical Crosslinking Assay

Usando um α-Bungarotoxina Binding Tag Site para Estudar GABA um receptor de membrana Localização e Tráfico

Testes anticorpos anti-RDL e Anti-mglutr1 em seções cerebrais de abelhas usando CRISPR-Cas9

Utilizando Receptores pHluorin-tag para monitorar subcelular Localização e Tráfico