Stereoinvestigator software 8

Anesthetized mice were transcardially perfused using PBS followed by 4% paraformaldehyde (PFA). Brains were extracted and post-fixed in 4% PFA overnight. Free-floating 50 µm vibratome (Leica, Wetzlar, Germany) sections were incubated overnight with anti-IBA1 (1:1000, 019-19741, Wako, Japan). Alexa Flour 488-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) were used at 1:200 for 2 h at room temperature. Nuclei were counterstained using 4′6-diamidino-2-phenylindole (DAPI, Roche, Basel, Switzerland) and sections were mounted on glass cover slips. Imaging was performed using the Leica TCS SP8 confocal laser scanning microscope (Leica, Wetzlar, Germany) and LAS AF image analysis software (Leica, Wetzlar, Germany). For DAB (3,3′-diaminobenzidine) staining, peroxidase-conjugated secondary antibodies (Dianova, Hamburg, Germany) were used. Images were captured using A Zeiss AxioImager I (Zeiss, Göttingen, Germany) and the Stereoinvestigator Software 8.0 (MicroBrightField, Magdeburg, Germany).

Stereological countings were performed on 30 μm coronal serial sections analyzing every sixth section for the SNpc and VTA. This was done using an oil immersion 63x objective, a counting frame of 50 x 50 μm, and a grid size of 100 x 100 μm, as described previously [27 (link)] using the optical fractionator method of the StereoInvestigator software 8.0 (MicroBrightField). To quantify lacZ positive and TH positive cells (Fig 1) we counted at least 2 representative 30 μm serial coronal sections. Venus positive and TH positive cells (Fig 2) were quantified in at least 3 representative 50 μm serial sagittal sections per mouse (every forth section) by applying a binary mask and the “Analyze Particles” function in ImageJ (NIH).

For the stereological analysis, a Zeiss AxioImager I (Zeiss, Göttingen, Germany) and StereoInvestigator Software 8.0 (MicroBrightField, Magdeburg, Germany) was used. To calculate the infarct volume and the number of surviving (NeuN‐positive) neurons in the infarct area, a series of brain sections of 60 µm in thickness was used: Every 360 µm was sampled, starting from the first section with an obvious infarct (as defined by the Neurotrace 435 staining) and ending with the last one with an obvious infarct. Then, the optical fractionator workflow provided by the software was used to extrapolate the sampled subvolumes and thus estimate the volume of the entire cell population. A virtual space called an optical dissector was used, and counting rules were followed to prevent overestimating. In each brain section, the infarcted area that was still present (infarct volume) and the infarcted area that was missing because of cyst formation after necrosis (ischemic core volume) were separately outlined. Then, the neuronal density within the infarcted volume and the neuronal density on the intact contralateral (control) side were estimated and the ratio between the two densities were calculated.