Tcs sp5 multiphoton laser scanning confocal microscope

Immunofluorescence was performed as previously described.⁷ The rats’ brains were fixed with 4% paraformaldehyde and then cut into 20‐mm sections. The following primary antibodies were used: anti‐NLRP3, 1:50, ab4207, Abcam; anti‐p65, 1:50, 10745‐1‐AP, Proteintech; anti‐Iba‐1, 1:200, ab5076, Abcam; anti‐GFAP, 1:50, A0237, AbClonal. Fluorescence‐labeled secondary antibodies were applied and 4′,6‐diamidino‐2‐phenylindole (DAPI, Invitrogen) was used to stain the nuclei. Samples were visualized using a TCS SP5 multiphoton laser scanning confocal microscope (Nikon, Tokyo, Japan).

Capillaries were identified, as described previously, by intravenous injection of 0.2 ml FITC-Dextran (50 mg/ml, Sigma) 10 min before animals were sacrificed24 (link). In brief, three coronal cryosections (20 μm) from each rat at bregma −0.2, −0.8, and −2.8 mm were analyzed by using a TCS SP5 multiphoton laser scanning confocal microscope (Nikon, Japan). Ten fields of view from each coronal section were collected from the IBZ after the left pMCAO. A threshold was applied to each digitized image to ensure that the number of FITC pixels reflected the original pattern of FITC-Dextran-perfusion. Data were presented as a percent ratio between the number of FITC after injection of antagomir-107 or agomir-107 into rat IBZ pixels and the antagomir control.