Cm1900 frozen slicer

Immunofluorescence staining was performed on frozen coronal sections of mouse brains or on primary microglia plated on cover slips. The mouse brains were fixed with 4% paraformaldehyde. After post-fixation and concentration gradient dehydration, the brains were cut into 10-μm-thick sections using a Leica CM1900 frozen slicer. The cells were fixed with 4% paraformaldehyde for 30 min. The brain sections and cell cover slips were washed three times with PBS and then incubated overnight at 4 °C in a humidified atmosphere with primary antibodies. The following primary antibodies were used: mouse anti-TLR4 (1:100; Santa Cruz, USA), rabbit anti-TLR4 (1:100; Santa Cruz, USA), rabbit anti-GPR30 (1:200; Abcam, England), goat anti-Iba1 (1:200; Novus, USA), rabbit anti-Iba1 (1:500; Wako, Japan), and mouse anti-NeuN (1:200; Millipore, USA). Then, the samples were incubated with mixtures of Alexa-488 (red, Invitrogen) or Alexa-594 (red, Santa Cruz) and Alexa-647 (green, Invitrogen)-conjugated donkey anti-goat or anti-rabbit and donkey anti-mouse secondary antibodies for 2 h in the dark at room temperature. Finally, the sections were photographed using an Olympus BX51 (Japan) fluorescence microscope. The average area of single microglial cells was measured by ImageJ software.

Nissl staining was applied to observe morphological changes in cells within the ischemic penumbra. The brains were perfused with cold 4% paraformaldehyde. Then, the brains were removed after post-fixation and dehydrated in 20 and 30% sucrose solutions. Subsequently, 10-μm-thick sections were prepared using the Leica CM1900 frozen slicer. The experimental steps were strictly performed according to the manufacturer’s manual of the Nissl staining kit (#G1430, Solarbio, China). The cells with large cell bodies, a rich cytoplasm, and one or two large, round nuclei were intact neurons, while the cells with shrunken cell bodies, condensed nuclei, dark cytoplasm, and empty vesicles were damaged neurons, as described in the manufacturer’s manual. Cells in five different fields were counted.