Animal experiments were performed in accordance with the Policy of Ethics and the Policy on the Use of Animals in Neuroscience Research as approved by the European Communities Council Directive 2010/63/EU of the European Parliament and of the Council of the European Union on the protection of animals used for scientific purposes.

1. Preparation of Instruments and Culture Media

1. For preparation of OHSC use the following set of instruments: two small scissors, two curved tweezers, one tweezer with a fine tip, three blades (two of size 11, one of size 15), three scalpel holders, round filter papers (diameter: 35 mm), agar, one razor blade, one unmodified Pasteur pipette, and one modified Pasteur pipette without a tip. Sterilize all materials in an autoclave before usage (Figure 1).
2. Weigh 5 g agar and dissolve it in 100 mL distilled water. Sterilize the solution for 20 min at 121.7 °C at 210.8 kPa in an autoclave. Distribute the 3 mL liquid agar solution in 60-mm Petri dishes using a sterile glass pipette, allow it to solidify for 5 h, cover with plastic paraffin film to avoid contamination and store at 4 °C until further use. Agar blocks are needed to stabilize the brains during the slicing procedure.
3. Media
   1. Make 200 mL of the preparation medium (pH 7.35) consisting of 198 mL minimal essential medium (MEM) and 2 mL L-glutamine solution (final concentration 2 mM). Prepare the solution on the day of media preparation and store it at 4 °C.
   2. Prepare 100 mL of the culture medium consisting of 49 mL MEM, 25 mL Hanks' balanced salt solutions (HBSS), 25 mL (v/v) normal horse serum (NHS), 1 mL L-glutamine solution (final concentration 2 mM), 100 µg insulin, 120 mg glucose, 10 mg streptomycin, 10,000 U penicillin, and 800 µg vitamin C as reported previously. Warm the medium (37 °C), adjust the pH value to pH 7.4 and sterile filter (0.2 µm pore size). Repeat the procedure (warming up, pH adjustment, etc.) every second day before changing the medium. Use the medium at the most for one week when stored at 4 °C.
4. Fill one 35-mm Petri dish with preparation medium to store the brain. Place two empty Petri dishes for collecting the tissue on a cooling pack in the working area.

2. Preparation and Slicing with a Vibratome

1. Use brains from 7-9 day old rats or 4-5 day old mice for the OHSC preparation according to Stoppini et al.⁴ After decapitation of animals, remove the skin from the skull with scissors.
2. Introduce the blade of a fine scissor into the foramen magnum and open the skull by cutting along the caudal (back) rostral (front) axis. Make two cuts perpendicular to the first one, so that the scissors point towards the left and right ear, respectively ("T-incision").
3. Open the skull carefully with fine forceps, paying attention not to injure the brain. Use a scalpel (blade size 11) to cut the most rostral part of the frontal pole and the cerebellum.
4. By means of a spatula, remove the brain and place it carefully in the Petri dish filled with preparation medium (Figure 2). Position the brain on the specimen holder and fix it with medical cyanoacrylate glue. Use the pieces of agar to assure mechanical stabilization.
5. Dissect the tissue horizontally in 350 µm thick OHSC using a sliding vibratome.
6. Evaluate the slices optically using a binocular microscope. Discard OHSC of low quality immediately. It is important to take only those slices with intact cytoarchitecture isolated from the middle part of the hippocampus (see Figures 3 and 4) between the dorsal and the ventral hippocampus.
7. Separate the hippocampal region and the entorhinal cortex using a scalpel (round blade size 15; Figure 3). The perforant pathway and entorhinal cortex must be preserved.
8. Six to eight OHSC are obtained from each brain. Transfer 2-3 slices into one cell culture insert (pore size 0.4 µm) and place it in one well of a 6-well culture dish containing 1 mL culture medium per well.
9. Incubate the 6-well dishes at 35 °C in a fully humidified atmosphere with 5% (v/v) CO₂ and change the cell culture medium every second day.
   NOTE: Conduct your experiments at 6 day in vitro (div). Inflammatory reactions associated with the slicing procedure disappear by day 6. At this stage, microglial cells show a ramified morphology again and synaptic connections have matured.

3. Evaluation of Tissue Quality

1. Fixation, labeling and visualization of degenerating neurons in OHSC
   1. After performing the experiments, incubate the OHSC with 5 µg/mL propidium iodide (PI) for 2 h prior to fixation, in order to stain the nuclei of degenerating neurons.
      CAUTION: PI is a suspected carcinogen, always wear personal protective equipment (PPE) such as gloves.
   2. Wash the OHSC with 0.1 M phosphate buffer (pH 7.4) and fix them with a 4% (v/v) solution of paraformaldehyde (PFA) for 24 h.
      CAUTION: PFA is a toxic and suspected carcinogen. Work under fume hood and wear PPE.
   3. Wash the OHSC in inserts with 1 mL PBS and use a rigger brush to separate slices from the membrane.
   4. Label the OHSC with IB₄ dye in a 24-well plate (Figure 5).
2. Conduction of tumor invasion experiments using fluorescently labeled single cells
   1. 24 h prior to the start of an experiment, label the tumor cells by using the fluorescent dye Carboxyfluorescein diacetate succinimidyl ester (CFDA SE).
   2. Detach and count the tumor cells using a Neubauer chamber.
   3. Resuspend the cells in medium, such that 10 µL of suspension contains the desired cell number (normally 50,000 or 100,000 cells).
   4. Apply 10 µL of the cell suspension onto the slice culture and allow the cells to invade for 3 days.
   5. At the end of the experiment, fix the slice cultures using 4% (v/v) PFA for 24 h, and label the co-cultures with PI for another 24 h to visualize the cytoarchitecture.
      CAUTION: PFA is a toxic and suspected carcinogen. Work under fume hood and wear PPE.
   6. Mount the co-cultures onto a cover slip for further analysis using the mounting media.

4. Evaluation of OHSC Experiments

Analyze the OHSC with a confocal laser scanning microscope (CLSM). For detection of PI labeled, degenerating neurons or the PI labeled cytoarchitecture use monochromatic light of the wavelength λ = 543 nm and an emission band pass filter for wavelengths λ = 585-615 nm. For CFDA labeled tumor cells or IB₄ microglia, use an excitation wavelength of λ = 488 nm. For both experimental types, record a z-stack with 2 µm thick optical slices and used for evaluation.