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

Neuroscience

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Summary

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.

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Fernandes, A., Mendes, N. D., Almeida, G. M., Nogueira, G. O., Machado, C. d., Horta-Junior, J. d., Assirati Junior, J. A., Garcia-Cairasco, N., Neder, L., Sebollela, A. Short-Term Free-Floating Slice Cultures from the Adult Human Brain. J. Vis. Exp. (153), e59845, doi:10.3791/59845 (2019).

Abstract

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.

Introduction

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.

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Protocol

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 Ribeirão 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 Ribeirão Preto Medical School, University of São Paulo, Brazil).

1. Sterilization of materials

NOTE: All material and solutions must be sterilized prior to use.

  1. Sterilize all surgical tools and vibratome slicing material (knife holder, specimen disk, buffer tray) in a dry sterilizing oven for 4 h at 180 °C.
  2. Sterilize temperature-sensitive material or equipment by UV or gamma irradiation.
  3. Sterilize media and solutions by autoclavation or filtration through 0.22 µm pore membranes.

2. Preparation of solutions

  1. Prepare 15-20 mL of transport solution: 50% v/v Hanks' 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 µg/mL gentamicin.
    NOTE: Transport solution must be refrigerated and oxygenated (bubbling with carbogen gas) for at least 20 min prior to sample collection.
  2. 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.
  3. 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 µg/mL amphotericin B.

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.

  1. 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.
  2. 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).
  3. Set the vibratome for slicing: section thickness of 200 µm, frequency of vibration of 100 Hz, and speed of slicing between 0.5-1.0 mm/s.
  4. 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.

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.

  1. 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.
  2. Collect and transport the specimen (Figure 1B) immediately to the lab. Submerse the specimen in cold transport solution (constantly bubbled with carbogen mixture).

5. Slicing

  1. 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).
  2. 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.
  3. 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).
  4. 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).
  5. 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.
  6. Lock the knife holder in place with the razor blade firmly fixed.
  7. The slicing solution must cover both the specimen and the blade, only then start slicing (Figure 1F).
  8. Cut the specimen into 200 µm 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.
  9. 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.

6. Culture

NOTE: Perform this step in a laminar flow cabinet under sterile environment.

  1. Add 600 µL of culture medium per well (in a 24 well plate) and incubate for at least 20 min at 36 °C and 5% CO2 prior to plating the slices.
  2. Plate one slice per well using a paintbrush (Figure 1G).
  3. If there are any unused wells in the plate, fill them with 400 µL of sterile water.
  4. Incubate the plate at 36 °C, 5% CO2.
    NOTE: First medium replacement must be done in between 8-16 h after plating depending on the size of the slice.
  5. 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 µL 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 µL.
  6. Remove 333 µL of the conditioned medium from each well and add 133 µL of fresh BDNF-supplemented medium.
  7. Repeat the process of medium replacement every 24 h by replacing one-third of the conditioned medium with fresh BDNF-supplemented medium.

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.

  1. 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.
    1. In the laminar flow cabinet, replace one-third of the medium with fresh medium.
    2. 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.
    3. Incubate the plate for 24 h at 36 °C, 5% CO2.
      NOTE: After H2O2 treatment (or other toxic stimulus of interest), the following steps describe cell viability determination by the MTT assay.
    4. Add 40 µL of MTT solution to a final concentration of 0.5 mg/mL to each well in the plate.
    5. Incubate the plate at 36 °C, 5% CO2 for 3 h.
    6. Wash the slices with phosphate-buffered saline (PBS) and transfer to a microtube.
    7. Carefully remove any remaining solution by pipetting.
    8. 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 °C) at this stage for later processing.
    9. Homogenize the slices in 200 µL of isopropanol/HCl using a motorized pestle.
    10. Centrifuge at room temperature (RT) for 2 min at 2600 x g.
    11. Collect the supernatant and measure the absorbance at 540 nm.
  2. 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 .
    1. At the flow cabinet, replace the culture medium by 300 µL of HBSS previously equilibrated to 36 °C.
    2. Replace the HBSS with 300 µL of fresh HBSS previously equilibrated to 36 °C.
    3. Incubate the plate at 36 °C, 5% CO2 for 15 min.
    4. Replace the HBSS with either the fresh HBSS or with 80 mM KCl depolarizing solution (both at 36 °C) and incubate at 36 °C, 5% CO2 for 15 min.
    5. Transfer the slices from the plate to microtubes. At this step, slices can be stored at -20 °C for later processing.
    6. Prepare tissue extracts in 150 µL 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 °C for 10 min at 16000 x g, collect supernatant, and determine total protein concentration using the Bradford method.
    7. Load 30 µg of total protein onto a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
    8. After electrophoresis, transfer the gel content to a nitrocellulose membrane.
    9. 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 °C. After washing, incubate for 2 h at RT with rabbit anti-ERK1/2 (1:1,000).
    10. Incubate with the appropriate HRP-conjugated secondary antibody at RT for 1 h.
    11. Reveal with the preferred HRP substrate.
  3. 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.
    1. Fixing, cryoprotecting and resectioning the slices
      1. Transfer the slices from the wells with culture medium to a new 24 well plate containing PBS.
      2. 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 °C.
      3. Carefully remove the PFA solution and add 1 mL of 30% sucrose solution. Incubate for 48 h at 4 °C.
      4. Set the freezing microtome to -40 °C.
      5. 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.
      6. Place each slice over a stretched plastic film and use a paintbrush to flat the tissue.
      7. 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.
      8. Let the slice rest for 5-10 min for proper freezing.
      9. Cut the slice into 30 µm sections.
      10. Transfer the 30 µm sections to a Petri dish containing PBS.
      11. Proceed to the histology protocol more adequate to the experimental design.
        NOTE: The 30 µm sections can be readily used for free-floating immunohistochemistry, mounted onto microscopy slides for further histology or stored in antifreeze solution at -20 °C.
    2. 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.
      1. Incubate the slices in blocking solution (e.g., 2% normal donkey serum in PBS) for 40 min.
      2. Incubate overnight with primary antibody under mild agitation at 4 °C.
      3. After washing with PBS, incubate for 120 min with biotinylated secondary antibody under mild agitation at RT.
      4. After washing with PBS, incubate at RT for 120 min with avidin-peroxidase conjugate (Table of Materials).
      5. Reveal with DAB + 0.04% nickel ammonium.
      6. 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.

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Representative Results

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 that tissue architecture is not significantly affected either by the surgical procedure/sample processing or by the short-term period in vitro. In accordance with previous findings, it was shown that NeuN immunoreactivity was not altered between DIV0 and DIV432. Based on these results, the free-floating culture format, associated to the reduced thickness of the slice to 200 µm (compared to the widely used 300-400 µm when membrane inserts are used), contributed to better diffusion of oxygen and nutrients to inner cell layers in slices, which has been previously demonstrated to be critical to the health of cultured slices39,40.

Quantification of cell death in slices is also a valuable approach in ex vivo models of neuropathologies, such as slice cultures (reviewed by Lossi et al.41). In a previous study, we used the MTT assay to determine cell death levels along the period in culture32. In addition to viability, that same study also showed that cultured slices (up to DIV4) preserved the capacity to release neurotransmitters upon KCl-induced depolarization32. Here, those findings were expanded by investigating the neuronal response to KCl-induced depolarization on ERK phosphorylation, a central kinase involved in processes such as synaptic plasticity and memory42,43. Interestingly, a clear increase in ERK phosphorylation was seen in KCl-treated slices at DIV4 (Figure 3A,B).

Finally, the response of slices at DIV4 to a toxic challenge was evaluated with a known oxidative stress inducer, H2O2. The rationale was that the extent of cell death should be proportional to the level of cell viability in the cultured slices. As shown in Figure 3C, exposing the slices to 300 mM H2O2 for 24 h led to a robust decrease in MTT reduction. Taken together, the massive cell death observed in DIV5 after the H2O2 challenge and the KCl-induced depolarization results indicate that the preserved general health of slices at DIV4 responds adequately to a toxic stimulus such as oxidative stress.

Figure 1
Figure 1: Sample collection, transport, slicing, and culturing of cortical tissue from adult humans. The procedure starts at the surgical room with collection of cortical tissue from temporal lobectomy for the treatment of pharmacoresistant epilepsy (A,B). (C) Tissue fragment (n = 1) is immediately transferred to a tube containing ice-cold oxygenated transport medium (see below). (D) In the lab, meninges are removed using fine ophthalmic tweezers. Excess liquid is dried using filter paper, and the fragment is superglued (E) to the vibratome specimen disk with the white matter facing down and pial surface facing up. (F) Using a commercial shaving razor, the specimen is cut into 200 µm slices that are collected with a delicate paintbrush and transferred back to the Petri dish for further trimming of excess white matter and loose ends (not shown) prior to (G) plating and culturing in a free-floating format. (H) Slices cultures are kept viable for several days and can be used in a variety of experimental protocols. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Morphological analysis of neural cells in slice cultures from adult human brain. Slices were fixed at the fourth day in vitro. (A,B,C) Representative steps of the sectioning procedure prior to immunohistochemistry. Tissue was digitally colored to improve visualization. (D) Normal distribution of neurons in cortical layers (Roman numerals). (E) Microglia and (F) astrocytes were also clearly observed (n = 1 slice per cell type labelling). All slices were obtained from tissue from a single donor. Scale bars = 100 μm. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Functional and cell viability assays in adult human brain slice cultures. Neuronal activity was evaluated in slices at day in vitro 5 (DIV5) by KCl-induced depolarization and consequent increase in ERK phosphorylation. (A) A representative immunoblot result obtained with homogenates from one slice. Bands corresponding to phospho ERK (Perk) and total ERK (tERK) are indicated. (B) Quantification of pERK/tERK ratio in three independent slices from a single human donor. (C) Hydrogen peroxide (H2O2) toxicity was evaluated by the MTT assay in slices at DIV 5. Optical density values obtained were normalized by the mass of each slice. Slices were challenged with H2O2 at the indicated concentrations (No H2O2; 30 mM H2O2; 300 mM H2O2) for 24 h. Images of representative slices after incubation with MTT are shown above the bars. Results are presented as average ± standard error from data obtained from three independent slices from a single donor. Please click here to view a larger version of this figure.

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Discussion

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 plasticity46,47, long-term plasticity48,49, neurotoxicity/neuroprotection10,11,12,13, cell therapy14, drug screening15,16,17, cancer50,51, genetics, and gene editing12,18,19,20.

The use of samples from adult human tissue is particularly important to understanding human neuropathologies, due to unique properties typical of the human brain lacking in slices produced from rodent nervous tissue52. Moreover, slice cultures from rodent brains are commonly prepared from neonatal, immature brains, which are highly plastic and contain migrating cells that may change the original slice cytoarchitecture to adapt to the in vitro environment26,53,54,55. Such plastic events lead to changes in circuitry that should be avoided when the goal is to mimic the in vivo condition, as stated by Ting et al.30: "We opted to focus on cultures of less than one week, where structural and functional properties are reasonably maintained with minimal perturbation, to be as comparable as possible to measurements obtained using gold-standard acute slice preparations". Therefore, although a method devoted to short-term culturing may initially be seen as limited, long-term culturing is not needed to produce relevant results in many experimental designs32,33,34,35,56,57.

Two critical steps of the protocol are the reduced thickness of the slices (200 µm) and supplementation of the culture medium with BDNF. In previous work32, we have shown preservation of cell viability up to 4 DIV and discussed the likely contribution of the reduction of slice thickness to 200 µm compared to the more often used 300-400 µm slices12,29. Basically, reducing slices thickness may contribute to a better oxygenation and nutrients uptake in free-floating slices, decreasing the chances of core hypoxia and neurodegeneration31,32,57,58,59,60,61. In addition, it is recommended to keep tissue in cold, oxygenated media from the surgical room to the slicing step at the vibratome, considering the high demand for O2 by the adult human central nervous. Supplementation of medium with BNDF has been previously seen to slightly increase viability in adult human brain slices cultured free-floating32, in line with recommendations for medium supplementation with neurotrophic factors by other authors62,63.

In conclusion, this protocol describes methods for preparing and running assays with short-term, free-floating slice cultures from adult human brains. This model should be amenable for investigations on the mechanisms of toxicity/neuroprotection relevant to age-associated brain diseases. The use of human resected tissue presents the advantage of preserving brain cytoarchitecture and local circuitry, adding translational power to obtained findings. Moreover, bursting the use of human-derived samples from neurosurgery in neuroscience may help reduce the need for animal experimentation. Although this protocol is centered around the use of tissue collected from patients submitted to neurosurgery for pharmacoresistant epilepsy treatment, it is suggested that tissue collected from other brain regions/conditions also be considered as sources for producing slice cultures in a simple and cost-effective way.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

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’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 Ribeirão Preto Medical School, University of São Paulo, who helped in various stages of the process.

Materials

Name Company Catalog Number Comments
2-Propanol Merck 1096341000
Acrylamide/Bis-Acrylamide, 30% solution Sigma Aldrich A3449 
Agarose Sigma Aldrich A9539
Ammonium persulfate Sigma A3678-25G
Amphotericin B Gibco 15290-018
Antibody anti-ERK 2 (rabbit) Santa Cruz Biotecnology sc-154 Dilution 1:1,000 in BSA 2.5%
Antibody anti-pERK (mouse) Santa Cruz Biotecnology sc-7383 Dilution 1:1,000 in BSA 2.5%
B27 Gibco 17504-044
BDNF Sigma Aldrich SRP3014
Bovine Serum Albumin Sigma Aldrich A7906
Bradford 1x Dye Reagent BioRad 500-0205
EDTA Sigma T3924 Used in RIPA buffer
Glucose Merck 108337
Glutamax Gibco 35050-061
Hank's Balanced Salts Sigma Aldrich H1387-10X1L
Hepes Sigma Aldrich H4034
Hydrochloric acid Merck 1003171000
Hydrogen Peroxide (H2O2) Vetec 194
Mouse IgG, HRP-linked whole Ab (anti-mouse) GE NA931-1ML
NaCl Merck 1064041000 Used in RIPA buffer
Neurobasal A Gibco 10888-022
Non-fat dry milk (Molico) Nestlé Used for membrane blocking
PBS Buffer pH 7,2 Laborclin 590338
Penicilin/Streptomicin Sigma Aldrich P4333
Potassium Chloride Merck 1049361000
Prime Western Blotting Detection Reagent GE RPN2232
Rabbit IgG, HRP-linked whole Ab (anti-rabbit) GE NA934-1ML
SDS Sigma L5750 Used in RIPA buffer
TEMED GE 17-1312-01
Thiazolyl Blue Tetrazolium Bromide (MTT) Sigma Aldrich M5655
Tris Sigma T-1378 Used in RIPA buffer
Triton x-100 Sigma X100 Used in RIPA buffer
Ultrapure Water Millipore Sterile water, derived from MiliQ water purification system
Equipment and Material
24-well plates Corning CL S3526 Flat Bottom with Lid
Amersham Potran Premium (nitrocellulose membrane)  GE 29047575
Carbogen Mixture White Martins 95% O2, 5% CO2
CO2 incubator New Brunswick Scientific CO-24 Incubation of slices 5% CO2, 36ºC
Microplate Reader Molecular Devices
Microtubes Greiner 001608 1,5mL microtube
Motorized pestle Kimble Chase
Plastic spoon Size of a dessert spoon
Razor Blade Bic Chrome Platinum, used in slicing with vibratome
Scalpel Blade Becton Dickinson (BD) Number 24 Used for slicing of tissue; recommended same size or smaller
Superglue (Loctite Super Bonder) Henkel Composition: Etilcianoacrilato; 2-Propenoic acid; 6,6'-di-terc-butil-2,2'-metilenodi-p-cresol; homopolymer
Vibratome  Leica 14047235612 - VT1000S
Name of Material/ Equipment for Immunohistochemistry
Antibody anti-NeuN (mouse) Millipore  MAB377 Dilution 1:1,000 in Phosphate Buffer
Antibody anti-GFAP (mouse) Merck MAB360 Dilution 1:1,000 in Phosphate Buffer
Antibody anti-Iba1 (rabbit) Abcam EPR16588 - ab178846 Dilution 1:2,000 in Phosphate Buffer
Biotinylated anti-mouse IgG Antibody (H+L) Vector BA-9200
DAB Sigma Aldrich D-9015
Entellan Merck 107960
Ethanol Merck 1.00983.1000
Gelatin Synth 00G1002.02.AE Used for coating slides
Microtome Leica SM2010R Equipped with Freezing Stage (BFS-10MP, Physiotemp), set to -40ºC
Normal Donkey Serum Jackson Immuno Research 017-000-121
Paraformaldehyde Sigma Aldrich 158127
Rabbit IgG, HRP-linked whole Ab (anti-rabbit) GE NA934-1ML
Slides (Star Frost) Knittel Glaser Gelatin coated slides
Sucrose Vetec 60REAVET017050
Vectastain ABC HRP Kit (Peroxidase, Standard) Vector PK-4000, Kit Standard
Xylene Synth 01X1001.01.BJ

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