This work is supported by FAPESP (Grant 25681-3/2017 to AS), CAPES (Post-Doctoral fellowship PNPD/INCT-HSM to A.F. and Pre-Doctoral fellowship to N.D.M.) and FAEPA. G.M.A. holds a Master&#8217;s fellowship from FAPESP (MS 2018/06614-4). N.G.C. holds a CNPq Research Fellowship. We thank the patients and their families for donating the resected tissues for this study. We would like to acknowledge the support of residents, nurses, technicians, and the CIREP team, from the Clinical Hospital at the Ribeir&#227;o Preto Medical School, University of S&#227;o Paulo, who helped in various stages of the process.

Live adult brain tissues were obtained from patients undergoing resective neurosurgery for the treatment of pharmacoresistant temporal lobe epilepsy (Figure 1A). All procedures were approved by the Ethics Committee from the Clinics Hospital at the Ribeir&#227;o Preto Medical School (17578/2015), and patients (or their legal responsible person) agreed and signed the informed consent terms. Collection of the tissue was done by the neurosurgery team at the Epilepsy Surgery Center (CIREP - Clinics Hospital at the Ribeir&#227;o Preto Medical School, University of S&#227;o Paulo, Brazil).
1. Sterilization of materials
NOTE: All material and solutions must be sterilized prior to use.
<ol>
	<li>Sterilize all surgical tools and vibratome slicing material (knife holder, specimen disk, buffer tray) in a dry sterilizing oven for 4 h at 180 &#176;C.</li>
	<li>Sterilize temperature-sensitive material or equipment by UV or gamma irradiation.</li>
	<li>Sterilize media and solutions by autoclavation or filtration through 0.22 &#181;m pore membranes.</li>
</ol>
2. Preparation of solutions
<ol>
	<li>Prepare 15-20 mL of transport solution: 50% v/v Hanks&#39;&#160;balanced salt solution (HBSS) pH 7.4, 50% v/v basal medium for maintenance of post-natal and adult brain neurons (Table of Materials), supplemented with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), 3 mg/mL glucose, and 33 &#181;g/mL gentamicin. 
	NOTE: Transport solution must be refrigerated and oxygenated (bubbling with carbogen gas) for at least 20 min prior to sample collection.</li>
	<li>Prepare 300 mL of slicing solution (HBSS supplemented with 10 mM HEPES and 3 mg/mL glucose) and cool it down in a freezer to the point of initial crystal formation.</li>
	<li>Prepare 20 mL of culture medium: basal medium for maintenance of post-natal and adult brain neurons (Table of Materials) supplemented with 1% L-glutamine derivative (Table of Materials), 2% supplement for neural culture (Table of Materials), 1% penicillin/streptomycin, and 0.25 &#181;g/mL amphotericin B.</li>
</ol>
3. Setting up the slicing apparatus
NOTE: This protocol is ideally performed with the assistance of a colleague due to the logistics of sample collection in the surgical room.
<ol>
	<li>In a bucket of salt-added ice, let the slicing solution rest under carbogen mixture bubbling (95% O2, 5% CO2) for at least 20 min prior to use.</li>
	<li>Prepare a block of 3% agarose (approximately 2 cm x 2 cm x 2 cm) and superglue it to the vibratome specimen disk in order to create additional mechanical support to the tissue sample during slicing (Figure 1E).</li>
	<li>Set the vibratome for slicing: section thickness of 200 &#181;m, frequency of vibration of 100 Hz, and speed of slicing between 0.5-1.0&#160;mm/s.</li>
	<li>Lock the vibratome buffer tray to the vibratome base and add ice to keep it refrigerated prior to receiving the slicing solution and the sample, and throughout the slicing procedure.</li>
</ol>
4. Sample collection
NOTE: In this protocol, human neocortical tissue was collected in the surgical room and transported to the laboratory.
CAUTION: When dealing with human samples, follow the appropriate safety protocols established by the Institution.
<ol>
	<li>Set up the transport apparatus (Figure 1C) that consists of: a portable gas cylinder with carbogen mixture connected to a pressure/flux valve that controls the gas output connected to a silicon tubing that connects gas output to the transport vessel; a transport vessel, usually a 50 mL conical centrifuge tube with perforated lid for gas input, containing the transport solution; and ice for sample cooling during transport.</li>
	<li>Collect and transport the specimen (Figure 1B) immediately to the lab. Submerse the specimen in cold transport solution (constantly bubbled with carbogen mixture).</li>
</ol>
5. Slicing
<ol>
	<li>Transfer the specimen to a Petri dish (100 mm x 20 mm) containing slicing solution and, with fine surgical tools, carefully remove as much as possible of the remaining meninges in the sample (Figure 1D).</li>
	<li>Choose the best specimen orientation for producing slices with the particular characteristics of the experimental design, and with a no. 24 scalpel blade, trim a flat surface to be the base glued to the specimen disk.</li>
	<li>Using a disposable plastic spoon and delicate paintbrushes, collect the fragment from the Petri dish and dry excess solution using filter paper (dry by capillarity and avoid touching the tissue fragment with paper).</li>
	<li>Using superglue, attach the tissue to the vibratome specimen disk until it is firmly adhered to the disk and in contact with the agarose block (Figure 1E).</li>
	<li>Place the vibratome specimen disk (with tissue properly attached) in the vibratome buffer tray filled with slicing solution that must be bubbling during the whole process.</li>
	<li>Lock the knife holder in place with the razor blade firmly fixed.</li>
	<li>The slicing solution must cover both the specimen and the blade, only then start slicing (Figure 1F).</li>
	<li>Cut the specimen into 200 &#181;m slices. 
	NOTE: Although some vibratomes cut the specimens automatically, the close observation and minor adjustments in slicing speed during the process may help producing better slices. Discard initial irregular slices.</li>
	<li>Transfer the slices from the buffer tray to a Petri dish with slicing solution and trim loose edges and excess white mater to a proportion of around 70% cortex/30% white matter.</li>
</ol>
6. Culture
NOTE: Perform this step in a laminar flow cabinet under sterile environment.
<ol>
	<li>Add 600 &#181;L of culture medium per well (in a 24 well plate) and incubate for at least 20 min at 36 &#176;C and 5% CO2 prior to plating the slices.</li>
	<li>Plate one slice per well using a paintbrush (Figure 1G).</li>
	<li>If there are any unused wells in the plate, fill them with 400 &#181;L of sterile water.</li>
	<li>Incubate the plate at 36 &#176;C, 5% CO2. 
	NOTE: First medium replacement must be done in between 8-16 h after plating depending on the size of the slice.</li>
	<li>Supplement 10 mL of the previously prepared culture medium with 50 ng/mL brain derived neurotrophic factor (BDNF). 
	NOTE: During the first 8-16 h, the slices are incubated in 600 &#181;L of medium to avoid nutrient deprivation and acidification, since medium consumption in this phase is accelerated. From the next step, the volume of medium per well is adjusted to 400 &#181;L.</li>
	<li>Remove 333 &#181;L of the conditioned medium from each well and add 133 &#181;L of fresh BDNF-supplemented medium.</li>
	<li>Repeat the process of medium replacement every 24 h by replacing one-third of the conditioned medium with fresh BDNF-supplemented medium.</li>
</ol>
7. Evaluating health, morphology, and function in cultured slices
NOTE: To induce cell death for the purpose of illustration in the representative results, some slices were submitted to a 24 h treatment with the oxidative stress inducer H2O2. Steps 7.1.2 and 7.1.3 describe use of H2O2 to induce cell death.
<ol>
	<li>Cell viability 
	NOTE: A simple, straightforward method to evaluate neurotoxic/neuroprotection in challenged slice cultures is the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay36, which measures the percentage of metabolic active cells under normal conditions compared to treated samples.
	<ol>
		<li>In the laminar flow cabinet, replace one-third of the medium with fresh medium.</li>
		<li>From a 30% H2O2 stock solution, add a volume of H2O2 per well to reach the intended final concentration (30 mM or 300 mM). 
		NOTE: H2O2 was added after the daily change of the medium to guarantee the proper supply of fresh nutrients prior to the challenge.</li>
		<li>Incubate the plate for 24 h at 36 &#176;C, 5% CO2. 
		NOTE: After H2O2 treatment (or other toxic stimulus of interest), the following steps describe cell viability determination by the MTT assay.</li>
		<li>Add 40 &#181;L of MTT solution to a final concentration of 0.5 mg/mL to each well in the plate.</li>
		<li>Incubate the plate at 36 &#176;C, 5% CO2 for 3 h.</li>
		<li>Wash the slices with phosphate-buffered saline (PBS) and transfer to a microtube.</li>
		<li>Carefully remove any remaining solution by pipetting.</li>
		<li>Weigh the microtubes containing the slices to determine the mass of each slice (this is key for normalizing the absorbance readings obtained). 
		NOTE: If needed, samples can be frozen (-20 &#176;C) at this stage for later processing.</li>
		<li>Homogenize the slices in 200 &#181;L of isopropanol/HCl using a motorized pestle.</li>
		<li>Centrifuge at room temperature (RT) for 2 min at 2600 x g.</li>
		<li>Collect the supernatant and measure the absorbance at 540 nm.</li>
	</ol>
	</li>
	<li>KCl-induced neuronal depolarization 
	NOTE: Phosphorylation of the mitogen activated protein kinase (MAPK) signaling cascade protein ERK, followed by western blotting, can be used for the quantification of the neuronal response to KCl-induced depolarization37 .
	<ol>
		<li>At the flow cabinet, replace the culture medium by 300 &#181;L of HBSS previously equilibrated to 36 &#176;C.</li>
		<li>Replace the HBSS with 300 &#181;L of fresh HBSS previously equilibrated to 36 &#176;C.</li>
		<li>Incubate the plate at 36 &#176;C, 5% CO2 for 15 min.</li>
		<li>Replace the HBSS with either the fresh HBSS or with 80 mM KCl depolarizing solution (both at 36 &#176;C) and incubate at 36 &#176;C, 5% CO2 for 15 min.</li>
		<li>Transfer the slices from the plate to microtubes. At this step, slices can be stored at -20 &#176;C for later processing.</li>
		<li>Prepare tissue extracts in 150 &#181;L of radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl; 150 mM NaCl; 1 mM EDTA; 1% nonionic surfactant; 0.1% sodium dodecyl sulfate; pH 7.5). Centrifuge extracts at 4 &#176;C for 10 min at 16000 x g, collect supernatant, and determine total protein concentration using the Bradford method.</li>
		<li>Load 30 &#181;g of total protein onto a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).</li>
		<li>After electrophoresis, transfer the gel content to a nitrocellulose membrane.</li>
		<li>After blocking the membrane with 5% non-fat dry milk in TBS plus 0.1% Tween, incubate with mouse anti-pERK (1:1,000) for 16 h at 4 &#176;C. After washing, incubate for 2 h at RT with rabbit anti-ERK1/2 (1:1,000).</li>
		<li>Incubate with the appropriate HRP-conjugated secondary antibody at RT for 1 h.</li>
		<li>Reveal with the preferred HRP substrate.</li>
	</ol>
	</li>
	<li>Morphological evaluation 
	NOTE: In addition to cell survival and functional evaluations, it is important to analyze tissue morphology. Be aware that resectioning the cultured slice is an important step to producing as much high-quality material as possible for morphological analysis.
	<ol>
		<li>Fixing, cryoprotecting and resectioning the slices
		<ol>
			<li>Transfer the slices from the wells with culture medium to a new 24 well plate containing PBS.</li>
			<li>Remove the PBS and add 1 mL of 4% paraformaldehyde (PFA). It is important that slices be kept flat prior to adding PFA. Incubate overnight at 4 &#176;C.</li>
			<li>Carefully remove the PFA solution and add 1 mL of 30% sucrose solution. Incubate for 48 h at 4 &#176;C.</li>
			<li>Set the freezing microtome to -40 &#176;C.</li>
			<li>Prepare a sucrose base on the microtome stage where the slices should be placed (Figure 2A). Let it freeze completely and carefully cut some of the frozen sucrose to produce a flat surface on which the slice will be placed.</li>
			<li>Place each slice over a stretched plastic film and use a paintbrush to flat the tissue.</li>
			<li>Transfer the stretched slice to the frozen sucrose base in one single move. 
			NOTE: It is not possible to move the slice once it is over the frozen sucrose base. Perform this transfer step carefully.</li>
			<li>Let the slice rest for 5-10 min for proper freezing.</li>
			<li>Cut the slice into 30 &#181;m sections.</li>
			<li>Transfer the 30 &#181;m sections to a Petri dish containing PBS.</li>
			<li>Proceed to the histology protocol more adequate to the experimental design. 
			NOTE: The 30 &#181;m sections can be readily used for free-floating immunohistochemistry, mounted onto microscopy slides for further histology or stored in antifreeze solution at -20 &#176;C.</li>
		</ol>
		</li>
		<li>Immunohistochemistry 
		NOTE: Immunohistochemistry and immunofluorescence standard protocols vary among labs. For a detailed version of the protocol used here, refer to Horta et al.38. Primary antibodies used for immunostainings presented in Figure 2 include neuronal nuclei (NeuN), healthy mature neuron marker, glial fibrillary acidic protein (GFAP), astorcytes marker, ionized calcium binding adapter (Iba-1), and microglia marker.
		<ol>
			<li>Incubate the slices in blocking solution (e.g., 2% normal donkey serum in PBS) for 40 min.</li>
			<li>Incubate overnight with primary antibody under mild agitation at 4 &#176;C.</li>
			<li>After washing with PBS, incubate for 120 min with biotinylated secondary antibody under mild agitation at RT.</li>
			<li>After washing with PBS, incubate at RT for 120 min with avidin-peroxidase conjugate (Table of Materials).</li>
			<li>Reveal with DAB + 0.04% nickel ammonium.</li>
			<li>Mount the stained sections on gelatin coated microscopy slides and let them air dry. Dehydrate in ethanol, diaphanize in xylene, finish with mounting medium (Table of Materials), cover with a coverslip, and image.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

This protocol for producing free-floating, short-term slice cultures is an alternative method for culturing adult human neocortical slices. Such a protocol for slice cultures may be amenable for studies on (but not restricted to) optogenetics1,44,45, electrophysiology2,3,4,5, short-term plasticity<sup ...

The use of human samples in research is unequivocally a great option to study human brain pathologies, and modern techniques have opened new ways for robust and ethical experimentation using patient-derived tissue. Methods like organotypic/slice cultures prepared from adult human brain have been increasingly used in paradigms such as optogenetics1, electrophysiology2,3,4,5, plasticity6,7,8,9, neurotoxicity/neuroprotection10,11,12,13, cell therapy14, drug screening15,16,17, genetics and gene editing12,18,19,20, among others, as a strategy for better understanding neurological diseases during adulthood.
The comprehension of mechanisms underlying human brain pathologies depends on experimental strategies that require a large number of subjects. Conversely, in the case of slice cultures, although access to human samples is still difficult, the possibility of generating up to 50 slices from a single cortical sample partially circumvents the requirement of recruiting multiple volunteers by increasing the number of replicates and performed assays per collected tissue21.
Several protocols for brain organotypic/slice cultures have been described, ranging from the classical oculo drafts22,23 to roller tube24,25,26, semi-permeable membranes interface27,28,29,30, and free-floating slices31,32. Depending on the particularities of an experimental design, each technique has its own advantages and disadvantages. Short-term, free-floating slices cultures from adult human brains is in some cases advantageous over the method used by Stoppini et al.27, if considering the fact that although long-term cell survival in vitro is usually a major concern when evaluating a culture method, in many experiments only short periods of time in culture are needed12,31,32,33,34,35. Under these conditions, the use of free-floating cultures presents the advantage of being simpler and more cost-effective, as well as more accurately resembling the original human tissue condition than slices kept in culture over 2-3 weeks.
Despite the potential of slice cultures to neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly from human subjects. This article describes a protocol to use collected brain tissue from living human donors submitted to resective brain surgery to prepare free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are detailed. This protocol has been proven valuable for analyzing viability and neuronal function in investigations on the mechanisms of neuropathologies linked to adulthood.

<table><tbody><tr><td>2-Propanol</td><td>Merck</td><td>1096341000</td><td></td></tr><tr><td>Acrylamide/Bis-Acrylamide, 30% solution</td><td>Sigma Aldrich</td><td>A3449&amp;nbsp;</td><td></td></tr><tr><td>Agarose</td><td>Sigma Aldrich</td><td>A9539</td><td></td></tr><tr><td>Ammonium persulfate</td><td>Sigma</td><td>A3678-25G</td><td></td></tr><tr><td>Amphotericin B</td><td>Gibco</td><td>15290-018</td><td></td></tr><tr><td>Antibody anti-ERK 2 (rabbit)</td><td>Santa Cruz Biotecnology</td><td>sc-154</td><td>Dilution 1:1,000 in BSA 2.5%</td></tr><tr><td>Antibody anti-pERK (mouse)</td><td>Santa Cruz Biotecnology</td><td>sc-7383</td><td>Dilution 1:1,000 in BSA 2.5%</td></tr><tr><td>B27</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>BDNF</td><td>Sigma Aldrich</td><td>SRP3014</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma Aldrich</td><td>A7906</td><td></td></tr><tr><td>Bradford 1x Dye Reagent</td><td>BioRad</td><td>500-0205</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>T3924</td><td>Used in RIPA buffer</td></tr><tr><td>Glucose</td><td>Merck</td><td>108337</td><td></td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050-061</td><td></td></tr><tr><td>Hank&#039;s Balanced Salts</td><td>Sigma Aldrich</td><td>H1387-10X1L</td><td></td></tr><tr><td>Hepes</td><td>Sigma Aldrich</td><td>H4034</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Merck</td><td>1003171000</td><td></td></tr><tr><td>Hydrogen Peroxide (H2O2)</td><td>Vetec</td><td>194</td><td></td></tr><tr><td>Mouse IgG, HRP-linked whole Ab (anti-mouse)</td><td>GE</td><td>NA931-1ML</td><td></td></tr><tr><td>NaCl</td><td>Merck</td><td>1064041000</td><td>Used in RIPA buffer</td></tr><tr><td>Neurobasal A</td><td>Gibco</td><td>10888-022</td><td></td></tr><tr><td>Non-fat dry milk (Molico)</td><td>Nestl&amp;eacute;</td><td></td><td>Used for membrane blocking</td></tr><tr><td>PBS Buffer pH 7,2</td><td>Laborclin</td><td>590338</td><td></td></tr><tr><td>Penicilin/Streptomicin</td><td>Sigma Aldrich</td><td>P4333</td><td></td></tr><tr><td>Potassium Chloride</td><td>Merck</td><td>1049361000</td><td></td></tr><tr><td>Prime Western Blotting Detection Reagent</td><td>GE</td><td>RPN2232</td><td></td></tr><tr><td>Rabbit IgG, HRP-linked whole Ab (anti-rabbit)</td><td>GE</td><td>NA934-1ML</td><td></td></tr><tr><td>SDS</td><td>Sigma</td><td>L5750</td><td>Used in RIPA buffer</td></tr><tr><td>TEMED</td><td>GE</td><td>17-1312-01</td><td></td></tr><tr><td>Thiazolyl Blue Tetrazolium Bromide (MTT)</td><td>Sigma Aldrich</td><td>M5655</td><td></td></tr><tr><td>Tris</td><td>Sigma</td><td>T-1378</td><td>Used in RIPA buffer</td></tr><tr><td>Triton x-100</td><td>Sigma</td><td>X100</td><td>Used in RIPA buffer</td></tr><tr><td>Ultrapure Water</td><td>Millipore</td><td></td><td>Sterile water, derived from MiliQ water purification system</td></tr><tr><td>&lt;strong&gt;Equipment and Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>24-well plates</td><td>Corning</td><td>CL S3526</td><td>Flat Bottom with Lid</td></tr><tr><td>Amersham Potran Premium (nitrocellulose membrane)&amp;nbsp;</td><td>GE</td><td>29047575</td><td></td></tr><tr><td>Carbogen Mixture</td><td>White Martins</td><td></td><td>95% O2, 5% CO2</td></tr><tr><td>CO2 incubator</td><td>New Brunswick Scientific</td><td>CO-24</td><td>Incubation of slices 5% CO2, 36&amp;ordm;C</td></tr><tr><td>Microplate Reader</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Microtubes</td><td>Greiner</td><td>001608</td><td>1,5mL microtube</td></tr><tr><td>Motorized pestle</td><td>Kimble Chase</td><td></td><td></td></tr><tr><td>Plastic spoon</td><td></td><td></td><td>Size of a dessert spoon</td></tr><tr><td>Razor Blade</td><td>Bic</td><td></td><td>Chrome Platinum, used in slicing with vibratome</td></tr><tr><td>Scalpel Blade</td><td>Becton Dickinson (BD)</td><td>Number 24</td><td>Used for slicing of tissue; recommended same size or smaller</td></tr><tr><td>Superglue (Loctite Super Bonder)</td><td>Henkel</td><td></td><td>Composition: Etilcianoacrilato; 2-Propenoic acid; 6,6&#039;-di-terc-butil-2,2&#039;-metilenodi-p-cresol; homopolymer</td></tr><tr><td>Vibratome&amp;nbsp;</td><td>Leica</td><td>14047235612 - VT1000S</td><td></td></tr><tr><td>&lt;strong&gt;Name of Material/ Equipment for Immunohistochemistry&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Antibody anti-NeuN (mouse)</td><td>Millipore&amp;nbsp;</td><td>MAB377</td><td>Dilution 1:1,000 in Phosphate Buffer</td></tr><tr><td>Antibody anti-GFAP (mouse)</td><td>Merck</td><td>MAB360</td><td>Dilution 1:1,000 in Phosphate Buffer</td></tr><tr><td>Antibody anti-Iba1 (rabbit)</td><td>Abcam</td><td>EPR16588 - ab178846</td><td>Dilution 1:2,000 in Phosphate Buffer</td></tr><tr><td>Biotinylated anti-mouse IgG Antibody (H+L)</td><td>Vector</td><td>BA-9200</td><td></td></tr><tr><td>DAB</td><td>Sigma Aldrich</td><td>D-9015</td><td></td></tr><tr><td>Entellan</td><td>Merck</td><td>107960</td><td></td></tr><tr><td>Ethanol</td><td>Merck</td><td>1.00983.1000</td><td></td></tr><tr><td>Gelatin</td><td>Synth</td><td>00G1002.02.AE</td><td>Used for coating slides</td></tr><tr><td>Microtome</td><td>Leica</td><td>SM2010R</td><td>Equipped with Freezing Stage (BFS-10MP, Physiotemp), set to -40&amp;ordm;C</td></tr><tr><td>Normal Donkey Serum</td><td>Jackson Immuno Research</td><td>017-000-121</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma Aldrich</td><td>158127</td><td></td></tr><tr><td>Rabbit IgG, HRP-linked whole Ab (anti-rabbit)</td><td>GE</td><td>NA934-1ML</td><td></td></tr><tr><td>Slides (Star Frost)</td><td>Knittel Glaser</td><td></td><td>Gelatin coated slides</td></tr><tr><td>Sucrose</td><td>Vetec</td><td>60REAVET017050</td><td></td></tr><tr><td>Vectastain ABC HRP Kit (Peroxidase, Standard)</td><td>Vector</td><td>PK-4000, Kit Standard</td><td></td></tr><tr><td>Xylene</td><td>Synth</td><td>01X1001.01.BJ</td><td></td></tr></tbody></table>

short-term free-floating slice cultures from the adult human brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A critical aspect to evaluate the quality and health of cultured slices is the presence and typical morphology of the expected neural cell types, neurons, and glial cells. The typical architecture of the human cortical lamination was observed in a slice at DIV4, revealed by neuronal immunolabeling (Figure 2D). In addition, the expected presence of microglia and astroglia (Figure 2B,C) was also observed. These results demonstrate...

Watch this Scientific Journal Video about Short-Term Free-Floating Slice Cultures from the Adult Human Brain at JoVE.com

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...

short-term-free-floating-slice-cultures-from-the-adult-human-brain

Research

JoVE Journal

Neuroscience

8.8K Views.  University of São Paulo. We presenteren een protocol voor te bereiden vrij zwevende slice culturen uit hersenweefsel verzameld van levende menselijke donoren voorgelegd aan resective hersenchirurgie. Deze cultuur is vatbaar voor het uitvoeren van biochemische en celbiologie testen of immunohistochemie. Het zal naar verwachting bijdragen aan de opheldering van het mechanisme dat ten grondslag ligt aan neurodegeneratie in geassocieerde hersenziekten.Het belangrijkste voordeel van deze techniek is dat het een een...

Video: Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Developmental Biology

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Biology

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of their normal in vivo interactions. Slices obtained from a variety of transgenic mouse lines or use of viral vectors for expression of fluorescently tagged proteins or reporters in wild type brain slices allow for high-resolution imaging by fluorescence microscopy. Although several methods have been developed for imaging brain slices, combining slice culture with the ability to perform repetitive high-resolution imaging of specific cells in live slices over long time periods has posed problems. This is especially true when viral vectors are used for expression of exogenous proteins since this is best done in a closed system to protect users and prevent cross contamination. Simple modifications made to the roller tube brain slice culture method that allow for repetitive high-resolution imaging of slices over many weeks in an enclosed system are reported. Culturing slices on photoetched coverslips permits the use of fiducial marks to rapidly and precisely reposition the stage to image the identical field over time before and after different treatments. Examples are shown for the use of this method combined with specific neuronal staining and expression to observe changes in hippocampal slice architecture, viral-mediated neuronal expression of fluorescent proteins, and the development of cofilin pathology, which was previously observed in the hippocampus of Alzheimer's disease (AD) in response to slice treatment with oligomers of amyloid-&#946; (A&#946;) peptide.

Presented here is a modified roller tube method for culturing and intermittent high-resolution imaging of rodent brain slices over many weeks with precise repositioning on photoetched coverslips. Neuronal viability and slice morphology are well maintained. Applications of this fully enclosed system using viruses for cell-type specific expression are provided.

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of ...

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of cellular and synaptic properties. Nevertheless, there is an ever increasing need to perform correlated analyses between different experimental modes in addition to slice electrophysiology, for example: immunohistochemistry, molecular biology, in vivo imaging or electrophysiological recording; to answer evermore complex questions of brain function. However, making meaningful conclusions from these various experimental approaches is not straightforward, as even within relatively well described brain structures, a high degree of sub-regional variation of cellular function exists. Nowhere is this better exemplified than in the CA1 of the hippocampus, which has well-defined dorso-ventral properties, based on cellular and molecular properties. Nevertheless, many published studies examine protein expression patterns or behaviorally correlated in vivo activity in the dorsal extent of the hippocampus; and explain findings mechanistically with cellular electrophysiology from the ventro-medial region. This is further confounded by the fact that many acute slice electrophysiological experiments are performed in juvenile animals, when other experimental modes are performed in more mature animals. To address these issues, this method incorporates transcardial perfusion of mature (&#62;60 day old rodents) with artificial cerebrospinal fluid followed by preparation of modified coronal slices including the septal pole of the dorsal hippocampus to record from CA1 pyramidal cells. This process leads to the generation of healthy acute slices of dorsal hippocampus allowing for slice-based cellular electrophysiological interrogation matched to other measures.

The purpose of this protocol is to describe a method to produce slices of the dorsal hippocampus for electrophysiological examination. This procedure employs perfusion with chilled ACSF prior to slice preparation with a near-coronal slicing angle which allows for preservation of healthy principal neurons.

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of ...

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 &#956;m to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...

Preparing Free-Floating Slice Cultures from an Adult Human Brain

Source: Fernandes, A., et al. <a href="https://app.jove.com/v/59845">Short-Term Free-Floating Slice Cultures from the Adult Human Brain.</a> J. Vis. Exp. (2019).
This video demonstrates a technique for preparing free-floating slice cultures from the adult human brain. Thin slices are generated from a brain section using a vibratome. These slices are then suspended in a culture medium supplemented with growth factors to maintain tissue viability.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Sterilization of materials
NOTE: ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

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