Dp70 digital camera and software

Mice (5/group) were anesthetized with isoflurane and sacrificed following behavior test. The whole brain tissues were extracted immediately and cut sagittally into hemispheres. Left hemispheres were fixed in ice-cold 4% PFA overnight at 4 °C and were then equilibrated in 30% sucrose. Next, 25-μm sagittal slices in the hippocampi were obtained with a freezing microtome. Sections were washed in cold PBS three times, blocked with 5% goat serum for 1 h, and incubated with Iba-1 antibody (1:500) and BNIP3 antibody (1:200) overnight at 4 °C.
BV2 microglial cells were cultivated on cell slides for staining. Cells were fixed by 4% paraformaldehyde for 30 min at 37 °C and then permeated cell membrane using 0.1% Triton X-100 for 5 min at room temperature. Subsequently, the cell slides were incubated with LC3 (1:500), BNIP3 (1:200), TOM20 (1:200), NLRP3 (1:500), or ASC (1:500) antibodies at 4 °C overnight. After incubating with secondary antibody for 1 h at room temperature, all the slices were counterstained with DAPI (Vector Laboratories, CA, USA), and photomicrographs were captured using an Olympus DP70 digital camera and software (Olympus, Japan) or a confocal microscope (Nikon, Japan). Samples were analyzed in a blinded manner using 3–5 fields randomly selected from each group. The co-localization analysis was performed by the ImageJ software.

For the BBB behavioral test, 21 rats were anesthetized as described above and perfused intracardially with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 15 min. After fixation, post-fixation was performed overnight in 4% paraformaldehyde. For fluorescence immunostaining, non-specific labeling was blocked with 0.1% BSA in 0.1% Triton X-100/PBS for 60 min. The following primary antibodies were used and incubated with the tissue overnight at 4 °C: mouse monoclonal anti-BrdU (1:100, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), rabbit polyclonal anti-nestin (1:200, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Then, the slides were incubated with secondary antibody (goat anti-mouse conjugated to Alexa Fluor 488 or goat anti-rabbit conjugated to Alexa Fluor 566: 1:500, Molecular Probes, Eugene, OR, USA) for 1 h. Specimens were analyzed using an Olympus BX51 microscope and DP70 digital camera and software (Olympus, Tokyo, Japan).

Paraffin-embedded renal sections (3-μm-thick) were stained with periodic acid-Schiff (PAS). The quantification of tubular vacuolization, casts and cysts were assessed in 20 non-overlapping high-power fields (X40). All images were acquired using an Olympus BX51 clinical microscope and DP70 digital camera and software (Olympus, Tokyo, Japan).