*3.6. Limitations*

There are several limitations that may be considered given our conclusions:

Firstly, the described interactions of DMSO in the concentrations used may have interfered with the neuroprotective effects of both tested substances. However, this may only account for the highest concentration of 2.0 Vol.% of DMSO and not for the lower concentrations, not showing significant effects on the outcome in control as well as OGD. Secondly, we did not use a specific imaging technique to depict calcium concentrations. By applying well-established concentrations of nimodipine and amiloride, we are confident that an effective channel blockade was achieved, preventing calcium influx via these ion channels. Thirdly, we planned smaller groups for vehicle controls and control groups from the beginning to minimise the number of required animals. Thus, some control groups have limited sample size compared to treatment groups, and further power would be eligible regarding our observation of a possible neuroprotective effect of DMSO at a concentration of 1.0 Vol.%. Lastly, we did not investigate the effect of nimodipine in 2.0 Vol.% DMSO. Hence, it is not possible to draw specific conclusions from our observation of enhanced damage of combined treatment with nimodipine and amiloride at 2.0 Vol.% DMSO.

#### **4. Materials and Methods**

#### *4.1. Mediums*

Preparation medium (Gey's balanced salt solution (Sigma-Aldrich, Munich, Germany), 5 mg/mL D-(+)Glucose (Roth, Karlsruhe, Germany)) [63] was used for initial slice manufacturing. The growth medium used for slice culturing consisted of 50% Eagle minimal essential medium with Earle's salts (Sigma-Aldrich), 25% Hank's balanced salt solution (Sigma-Aldrich), 25% heat inactive horse serum (Sigma-Aldrich) with additional 5 mg/mL D-(+)Glucose (Roth, Karlsruhe, Germany), 1 Vol.% antibiotic/antimycotic solution [penicillin G GIBCO™, 10,000 units/mL, streptomycin sulphate 10 mg/mL, amphotericin B 25 μg/mL] (Thermo Fisher Scientific, Waltham, MA, USA), 5 μL/mL medium L-Glutamine solution (Sigma-Aldrich) and 10 μL/mL HEPES buffer solution (Sigma-Aldrich) [63]. For experiments, the experimental medium (75% Eagle minimal essential medium with Earle's salts (Sigma-Aldrich), 25% Hank's balanced salt solution (Sigma-Aldrich) with additional, 5 mg/mL D-(+)Glucose (Roth, Karlsruhe, Germany), 1 Vol.% antibiotic/antimycotic solution [penicillin G GIBCO™, 10,000 units/mL, streptomycin sulphate 10 mg/mL, amphotericin B 25 μg/mL] (Thermo Fisher Scientific, Waltham, MA, USA), 5 μL/mL L-Glutamine solution (Sigma-Aldrich) and 10 μL/mL HEPES buffer solution (Sigma-Aldrich)) or OGD medium (75% Eagle minimal essential medium with Earle's salts (Sigma-Aldrich), 25% Hank's balanced salt solution (Sigma-Aldrich) with additional 1 Vol.% antibiotic/antimycotic solution [penicillin G GIBCO™, 10,000 units/mL, streptomycin sulphate 10 mg/mL, amphotericin B 25 μg/mL] (Thermo Fisher Scientific, Waltham, MA, USA), 5 μL/mL L-Glutamine solution (Sigma-Aldrich) and 10 μL/mL HEPES buffer solution (Sigma-Aldrich)) were used, respectively. In essence, OGD medium is simply experimental medium without D-(+)Glucose.

#### *4.2. Slice Preparation and Cultivation*

The experiments in this article were strictly conducted according to institutional and governmental guidelines (TierSchG) with institutional permission by the animal protection representative of the Institute of Animal Research at the RWTH Aachen University Hospital and the local institutional committee (LANUV North Rhine-Westphalia, TV-11141A4). After decapitation of 4–7-day-old mice pups (C57BL/6N from Charles Rivers Laboratories, Sulzfeld, Germany and from Janvier Labs, La Rochelle, France, *n* = 138), their brains were extracted and instantaneously immersed into ice-cold preparation medium. The hippocampus slices were prepared using an already established method [58,63]. In brief, the brains were sagittally divided in half, and the frontal pole, as well as the cerebellum, was resected. Using a McIlwain Tissue Chopper (The Mickle Laboratory Engineering Co. ltd. [Now: Cavey Laboratory Engineering Co. ltd], Gomshall, UK), the brains were sliced into 400 μM thick slices from which the hippocampus was then carefully dissected. The hippocampus slices were then transferred onto MilliCell tissue culture inserts (MilliCell-CM, Millipore Corporation, Billerica, MA, USA) placed in 1 mL growth medium. Slices were cultivated at 37 ◦C and 5% CO2 for 14 days with the growth medium exchanged one day after the preparation and every following third day. On average, 9.1 slices per pup were prepared. Slices of one animal were allocated in two wells, which were then randomly allocated to the experimental groups to avoid allocation of slices of the same animal to only one experimental group. Overview in Figure 6a.

**Figure 6.** Experimental design. (**a**) Slice preparation, culturing for 14 days and baseline propidium iodide (PI) imaging. (**b**) OGD experiments with overview of experimental groups. (**c**) Incubation for 72 h after OGD, PI imaging for cell death assessment at 72 h, analysis of experimental groups. Created with BioRender.com.
