Superfrost plus adhesion microscope slides

GFP-expressing B. divergens in vitro cultures were smeared, air-dried and fixed with 4% paraformaldehyde and 0.075% glutaraldehyde in PBS for 30 min at RT on Superfrost Plus™ Adhesion Microscope Slides (Thermo Fisher Scientific). After fixation, the samples were washed with PBS (3 × 5 min) and incubated with 0.01% Triton X-100 (Merck) diluted PBS for 30 min at RT to permeabilize cell membranes. Anti-GFP Polyclonal Antibody conjugated with Alexa Fluor™ 488 (Thermofisher) was diluted 100× in 0.01% Triton X-100 and applied to samples for 1 hour at RT. Subsequently, the samples were washed in PBS (3 × 5 min) and stained with 300 nM DAPI diluted in PBS for 10 min at RT. After another round of washing with PBS (3 × 5 minutes), the samples were mounted in DABCO (Merck). GFP signal was examined by BX53F fluorescence microscope (Olympus) and processed in Fiji (ImageJ) software.

Cross‐sectional 14‐μm‐thick vastus lateralis cryosections were cut consecutively at −20°C, transferred to SuperFrost Plus adhesion microscope slides (Thermo Fisher Scientific Ltd., Loughborough, UK) and fixed by immersion into cold 2% Zamboni fixative (Newcomer Supply, Middleton, WI, USA), supplemented with 0.1% glutaraldehyde, for 1 h. Intramyocellular lipid stores were determined using the BODIPY 493/503 method.²³ Acquired images were used for the quantification of LD count, LD size and IMCL content in pre‐clamp muscle biopsy samples using the Fiji software package.²⁴

For histological studies, a separate cohort of mice underwent the same MCAO or sham surgery at 12 weeks of age and were sacrificed at 1-, 4-, 12-, 24-, 36- and 48-weeks post-surgery. After an overdose of Isoflurane, mice were transcardially perfused with cold 0.1 M phosphate buffer solution (PBS; 320 mM Na₂HPO₄ anhydrous, 90 mM NaH₂ PO₄.H₂O, pH 7.4) followed by 4% paraformaldehyde (PFA; w/v in 0.1 M PBS, pH 7.4). Brains were removed and post-fixed in 4% PFA overnight at 4 °C. Brains were cryoprotected with 30% sucrose, frozen with dry ice and stored at −80 °C until sectioned. Twenty-micron sections were prepared in a 1:12 series using a cryostat (CM1950, Leica Biosystems, Vic., Australia) and thaw-mounted on Superfrost Plus Adhesion microscope slides (ThermoFisher Scientific, Waltham, MA, USA). Sections were left to dry at room temperature overnight and then stored at −80 °C.

Before cryosectioning, 4% PFA-fixed slices were kept in 30% sucrose at 4 °C overnight for cryoprotection. The slices were cut out of the membrane insert with a piece of membrane below and mounted on the cryostat stage using Tissue-Tek® O.C.T. Compound (Sakura), with the slice surface facing downwards. Once the OCT had solidified, the membrane was carefully removed and an extra layer of OCT was added on top of the slice and left to freeze completely. The OHSCs were then cut on a cryostat (Leica, #CM1900) at a thickness of 10 μm and a temperature of − 20 °C. The sections were collected on Superfrost Plus Adhesion Microscope Slides (ThermoFisher) and stored at − 20 °C. Immunostaining was processed on the slides as previously described for the whole mount using an antibody against pS129 (D1R1R, rabbit mAb #23706S, Cell Signaling, 1:1000). During incubations, slides were kept in a humidity chamber with a hydrophobic pen barrier drawn around the tissue to prevent it from drying out. After the final washing step, the slides were mounted using glycerol gelatin aqueous slide mounting medium (Sigma, #GG1).

The histopathological evaluation of each ACT was performed as described previously.5 Formalin‐fixed paraffin‐embedded tissues were cut in 4 μm thick sections on Superfrost Plus Adhesion Microscope Slides (Thermo Fisher Scientific, Breda, The Netherlands). The tissue sections were stained with haematoxylin and eosin, and immunohistochemical staining for Ki67 (MIB‐1 clone, M7240, Dako, Agilent, Amstelveen, The Netherlands) was performed as described previously.5 All ACTs were evaluated by two observers. For each ACT, the Utrecht score was calculated: the Ki67 proliferation index +4 if ≥33% of cells have clear/vacuolated cytoplasm, and + 3 if necrosis was present. The ACTs were classified according to their Utrecht score as low risk of recurrence tumours (LRTs; < 6 in Utrecht score) or moderate‐high risk of recurrence tumours (MHRTs; ≥ 6 in Utrecht score).

For cryosectioning, brains were mounted on the cryostat stage (Leica) using Tissue-Tek® O.C.T. Compound (Sakura). After the OCT solidified, the brain was sliced at a thickness of 10–12 μm at -20°C. Sections were collected on Superfrost Plus Adhesion Microscope Slides (ThermoFisher) and subsequently processed for immunostaining. Tissue was permeabilized in 0.5% Triton X-100 followed by blocking with 10% BSA, for 45 minutes each at room temperature (RT) with gentle shaking. For primary antibody incubation, rabbit monoclonal pS129-α-syn (D1R1R, #23706, Cell Signaling Technology, 1:1000, RRID: AB_279886) was prepared in 5% BSA, and incubated overnight at 4°C. Slides were washed 3x 20 minutes in TBS + 0.03% Triton X-100, and then incubated with anti-rabbit Alexa Fluor 488 (Invitrogen, A11008, 1:2000) and DAPI (TH.GEYER, 5 μg/mL) for nuclear staining in 5% BSA for 2 hours at RT, protected from light. During antibody incubations, slides were kept in a humidity chamber with a hydrophobic barrier around the tissue to prevent them from the drying out. After the final washing step, the slides were mounted using DAKO fluorescent mounting medium (DAKO, #S3023). The edges of the coverslip were sealed using nail polish.