Dmire2 inverted fluorescent microscope

In order to determine the microcapsule membrane molecular weight cutoff (MWCO), FITC-dextrans were employed (Mw 10, 20, 40, 70, and 150 kDa). A volume of 10 μL of microcapsule suspension (≈170 capsules) was washed and a 0.5 mg/mL FITC-dextran solution was added. Samples were incubated at room temperature for 24 h and observed by confocal microscopy (Leica TCS SP2 AOBS Spectral Confocal Scanner (Buffalo, NY) mounted on a Leica DM IRE2 inverted fluorescent microscope, Wetzlar, Germany). Micrographs were analyzed using ImageJ. Equal area squares were defined and the relative intensity of 20 microcapsules and 20 background areas was determined to obtain the dextran diffusion percentage. Per study group four independent samples were assayed.

Brain sections were labeled with DeadEnd Fluorometric TUNEL System (Promega, Madison, WI, USA) or in situ cell death detection kit by DAB staining (Roche, Basel, Switzerland), following the manufacturer's manual as described previously.^{8 (link)} The sections were observed on a Leica DMIRE2 inverted fluorescent microscope.

The spinal cord was fixed with 4 % paraformaldehyde and then was embedded in paraffin using a Leica APS300. Five-micron cross sections were prepared, and antigen retrieval was performed by boiling sections in sodium citrate buffer (10 mM sodium citrate, 0.05 % Tween 20, pH 6.0) for 30 min. Anti-NLRP1 (Abcam), anti-HO-1 (Santa Cruz Biotechnology), and anti-MAP2 (Millipore) antibody were used for staining according to the manufacturer’s instructions. Alexa Fluor 488 donkey anti-rabbit IgG and Alexa Fluor 594 chicken anti-goat IgG (both from Invitrogen) were used as secondary antibodies. The sections were observed on a Leica DMIRE2 inverted fluorescent microscope.