Organotypic Slice Culture

2.3.4.1. Materials

2.3.4.1.1. Chemicals and Reagents

Product Company Cat. No.

5-Fluoro-2′-deoxyuridine Sigma F0503

BME (Basal Medium Eagle without Glutamin) Gibco 41010026

Cytosine β-D-arabinofuranoside hydrochloride (Ara-C) Sigma C6645

Glucose x H2O Fluka 49159

Glutamax (200mM) Gibco 35050038

Hank's Balanced Salt Solution , no Ca²⁺, no Mg²⁺

(HBSS) Gibco 14170-088

Heat-inactivated horse serum (HS) Gibco 26050-088

Kynurenic Acid Tocris 223

Millex Syringe Filter Units, 0.22 µm Gibco SLGP033RS

Millicell Cell Culture Inserts Millipore PICM03050

Millipore confetti Millipore FHLC04700

Minimum Essential Medium (MEM) Gibco 11700-077

razor blades Gillette

Uridine Sigma U3750

2.3.4.1.2. Equipment

2.3.4.1.3. Media and buffer 2xMEM (9.39g / 500ml ddH20)

Culture Medium (22.44ml ddH20, 25 ml 2xMEM, 25 ml BME, 1 ml Glutamax, 1.56ml Glucose 40%, 25 ml HS )

Cutting solution (pH 7.4; 97 ml HBSS, 2.5 ml Glucose 20%, 1 ml 100mM Kynurenic Acid) Inhibitor Mix (50 ml Culture Medium, 5µl of Ara-C, 5µl Uridine, 5µl 5-Fluoro-2′-deoxyuridine)

Product Company

McIllwain tissue chopper The Mickle Engineering Company Ltd, UK

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2.3.1.3. Methods

2.3.1.3.1. Preparation of organotypic hippocampal slice cultures

The protocol for the preparation of organotypic hippocampal slice cultures was based on the “interface method” introduced by Stoppini and colleagues in 1991 (Kerr et al., 2008;

Stoppini et al., 1991). Control and knockout mice were taken from the same mouse colony and from the same litters. For E18 preparations, the mothers were anesthetized, decapitated and the pups taken out by cesarean section. E18-P0 pups were then decapitated and the brains were placed into Cutting Solution. The hippocampi, still attached to the entorhinal cortex, were removed and transferred with a sterile Pasteur pipette onto the Tissue Culture Stage under a preparation hood. Excess liquid was removed with sterile autoclaved tissues. Slices of 300 μm thickness were cut with a McIllwain tissue chopper perpendicular to the longitudinal axis of the hippocampus. Then the stage was flooded with Cutting Solution and the slices were transferred in liquid with a plastic Pasteur pipette into a plastic dish. Only undamaged slices were then placed onto small membrane pieces (confetti) on top of the microporous cell culture membrane.

Usually, maximally four hippocampal slices were cultured on each cell culture insert.

Slices were maintained with culture medium. The medium was first changed 24h after preparation and then 2-3 times per week. Between 3-5 days in-vitro (DIV) 100 μM cytosine arabinoside, 100 μM uridine and 100 μM 5-fluorodeoxyuridine were added for 24h to the culture. The slices were maintained at 37°C and in 5% CO2 for 2-4 weeks.

High-pressure-freezing fixation for electron tomography was performed between 4 and 5 weeks in vitro. The animals were genotyped by using tail DNA and subsequent PCR analysis. In addition, for most of the experiments, the rest of the brain was frozen in liquid nitrogen and kept for Western-Blot analysis of the genotype if necessary.

2.4. Biochemistry

Amersham HyperfilmTM ECL GE Healthcare 28906837

Ammonium persulfate (APS) Sigma-Aldrich A-6761

Aprotinin Roth A162.3

Bio-Rad Protein Assay Dye Reagent BIO-RAD 500-0006

Bromophenol Blue Pierce 20730

Cell scraper Nunc 179693

Dithiothreitol (DTT) Boehringer Ingelheim 708992

ECL Reagent (Kit) Amersham, GE-Healthcare RPN2106V1

and -2 Ethylenediaminetetraacetic Acid Trisodium Salt (EDTA) Merck 1.084.180.250

Glacial acetic acid Merck 1.000.632.511

Glycine Sigma-Aldrich 33226

Goat Serum Gibcro, Invitrogen 16210-072

IGEPAL Sigma-Aldrich I3021

Instant Milk Powder Granovita GmbH

Leupeptin Pepta Nova 4041

MemCodeTM Reversible Protein Stain Kit Thermo Scientific 24580

Methanol J.T. Baker 8402

N,N,N',N'-Tetramethyl-ethylenediamine (TEMED) Serva 35925 N-2-Hydroxyethylpiperazine- N'-2-ethane sulfonic acid

(HEPES) Biomol 5288

PageRulerTM Prestained Protein ladder Fermentas SM0671

Phenylmethylsulfonylfluorid Sigma-Aldrich P7626

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2.4.1.2. Equipment

Product Company

Bio-Rad mini gel electrophoresis system (protean II) Bio Rad

BioScience Ultrospec3100pro Amersham

Blotting system, TE22 Mighty Snall tank transfer Hoefer

POTTER'S Homogenizer B.Braun

2.4.1.3. Media and buffer

Homogenization Buffer: (320mM sucrose, 5mM HEPES (pH7.4), 1mM EDTA, 0.1µM Aprotinin, 50µM Leupeptin, 0.2mM PMSF)

Lysis Buffer: (320mM Sucrose, 10mM Tris-HCl (pH 7.5), 5mM HEPES (pH7.4), 150mM NaCl, 1mM EDTA, 1mM EGTA, 0.1% IGEPAL, 0.2µM Aprotinin, 20µM Leupeptin, 1mM PMSF)

3x SDS-sample buffer: (10% SDS, 140mM Tris-HCL (pH 6.8), 3mM EDTA, 30% Sucrose, 100mM DTT, 0.1 % bromophenolblue,150 mM DTT)

Upper stacking gel (5 % AMBA, 125 mM Tris-HCL (pH 6.8), 0.1% SDS, 0.05% APS, 0.005%

TEMED)

Lower resolving gel (7-15% AMBA, 325 mM Tris-HCL (pH 8.8), 0.1% SDS, 0.05% APS, 0.005%

TEMED)

Running buffer(25mM Tris-HCl, 250 mM Glycine, 0.1% SDS, pH 8.8) Transfer Buffer:(25mM Tris-Base, 190 mM Glycine, 20 % Methanol) TBS:(10mM Tris-HCl, 150 mM NaCl, pH 7.5)

Blocking buffer:(5% milk powder, 5% Goat Serum, 9.1/ Tween 20 in TBS) Washing Buffer(0.1% Tween 20 in TBS)

Ponceau Red:(0.1% Ponceau, 5% glacial acetic acid in ddH20)

2.4.1.4. Antibodies

Product / concentration Company / Reference Cat. No.

anti-Munc13-1 (affinity purified rabbit polyclonal, N-terminal #40); 1:1000

(Varoqueaux et al. 2005;

Cooper et al. 2012) anti-Munc13-1 (affinity purified rabbit polyclonal,

C-terminal #N395); 1:250 (Betz et al., 1997)

anti-ß-Tubulin (mouse monoclonal) 1:5000 Sigma TUB 2.1

anti-Synaptophysin (purified mouse monoclonal)

1:10000 Synaptic Systems 101 011

Secondary goat anti-mouse and anti-rabbit affinity purified antibodies conjugated to horse radish peroxidase (HRP) (Bio Rad) were used in this study at a concentration of 1:10000.

2.4.2. Methods

2.4.2.1. Mouse Brain Homogenate

P0 mice were killed by decapitation and the brains were removed and each transferred into 1 ml ice-cold Homogenization buffer. The brain tissue was homogenized at 900 rpm with a glas/Teflon homogenizer at 4°C and collected for Western Blot analysis.

2.4.2.2. Cell Culture Homogenization

To check for changes in protein levels of lentiviral infected continental mouse hippocampal neuron cultures, neurons were washed twice with PBS and then incubated for 10min on ice in Lysis buffer. Samples were transferred to Eppendorf cups, centrifuged at 900x gmax for 10 minutes and the supernatant was collected and used for Western Blot analysis.

2.4.2.3. Preparation of Proteins Samples for SDS Electrophoresis

The protein concentration was determined using the Bradford Assay, measuring the absorbance of the sample at 595 nm wavelength with a BioScience Ultrospec3100pro spectrophotometer (Bradford, 1976). For a detailed protocol refer to the “Bio-Rad Protein Assay” manual. For all the biochemical experiments, samples were standardized to the same concentration in Homogenization- or Lysis buffer. The protein samples were then dissolved in SDS sample buffer to a 1x final concentration and boiled at 95°C for 5 minutes.

2.4.2.4. SDS-PAGE (Laemmli 1970) and Western Blotting (Towbin 1979;

Burnette 1981)

In order to separate proteins based on their molecular seize, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed under denaturing

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Im Dokument Molecular and Morphological Correlates of Synaptic Vesicle Priming (Seite 59-64)