C9600 nanozoomer

Coronal sections of 3 μm thickness were prepared from mouse brains injected with GBM cells. The sections were dried overnight at 55°C. Prior to staining, tissues were deparaffinized in xylol and rehydrated in graded series of ethanol. Antigen retrieval was required for Ki-67, MMP-9 and Nestin through microwave pretreatment with Tris/EDTA, Tris/HCl or citrate buffer, respectively. Subsequently endogenous peroxidase was blocked for 30 minutes using 0.3% H₂O₂ at room termperature (RT). Then slides were incubated with primary antibodies diluted in 1% BSA/PBS for 1 hour at RT. Antibodies used were GFAP (Z0334, Dako) diluted 1:250, Ki-67 (MIB-1, Dako) diluted 1:300, MMP-9 (C-20, Santa Cruz Biotechnology INC) diluted 1:50, and Nestin (10C2, Santa Cruz Biotechnology INC) diluted 1:100. Secondary and tertiary antibodies were diluted in 1% BSA/PBS and supplemented with 1% AB-serum and incubated for 30 minutes at RT. Staining was visualized by 3,3’-diaminobenzidine incubation for 10 minutes and sections were counterstained with haematoxylin and mounted. Images were acquired using a C9600 NanoZoomer (Hamamatsu Photonics KK, Hamamatsu City, Japan).

Slices were fixed in formalin (4%) at 4°C for 24 h, after which they were dehydrated in graded ethanol baths, cleared in xylene, and embedded horizontally in paraffin. Before staining, sections (4 μm) were deparaffinized in xylene and rehydrated in graded ethanol baths. Tissue morphology was investigated with a routine hematoxylin and eosin (H&E) staining and fibrillar COL1 and COL3 networks were visualized using a Picro Sirius Red Stain Kit (Abcam). High-resolution digital data was then obtained by scanning stained sections with a C9600 NanoZoomer (Hamamatsu Photonics, Hamamatsu, Japan). Semi-quantitative tissue damage scores were assigned to H&E stained sections using our previously published scoring system (23 (link)). The extent of collagen deposition was estimated by visual inspection of picrosirius red-stained sections using unpolarized light.

Perfusate and tissue samples were obtained at standardized timepoints and from standardized locations (Figures S1 and S2 [30 (link)] and Table S2). Perfusate samples were collected, centrifuged (1800 rpm for 10 min at 4 °C) and stored at −80 °C until further analysis. For the sevoflurane measurements, perfusate samples were collected and centrifuged (1800 rpm for 10 min at 4 °C), and 1 mL was pipetted into a 10 mL headspace tube, which was immediately sealed with a Teflon cap and stored at −80 °C [30 (link)]. Tissue samples were divided into 5 parts. Samples for wet weight/dry weight (WW/DW) measurement were stored at 4 °C, weighed the next day and then dried in an Eppendorf oven (Avantor, VWR, Analog Heatblock, Amsterdam, The Netherlands) for 24 h at 100 °C before they were weighed again. A second part of the tissue was stored in 4% neutral buffered formalin, cleared in xylene and embedded in paraffin. Sections of 4 μm were cut and stained with hematoxylin and eosin. High-resolution images of the sections were taken with a C9600 NanoZoomer (Hamamatsu Photonics, Hamamatsu, Japan) and assessed by the Department of Pathology, University Medical Center Groningen. Tissue samples for mRNA analysis were stored at −80 °C.