Rabbit anti cd3

Immunostaining was performed on free‐floating 25‐μm sections. Sections were incubated in the following primary antibodies overnight at 4°C: rabbit anti‐Iba1 (Wako), rabbit anti‐CD3 (Abcam), rabbit anti‐myelin basic protein (MBP; Abcam), mouse anti‐nonphosphorylated neurofilament H (SMI32; Calbiochem). Images were captured using a fluorescence microscope (Leica). Immuno‐positive cell counts were presented as the mean number of cells per square millimeter. Three randomly selected microscopic fields within the cortex, striatum, lateral corpus callosum or external capsule on each of three consecutive sections were analyzed for each brain by a blinded investigator. White matter injury was expressed as the mean ratio of SMI32 to MBP immunostaining. The lesion zones of cortex in dMCAO models were studied in more details using Western blot. Myelin basic protein (MBP), oligodendrocyte transcription factor (Olig‐2), CNPase, and oligodendrocyte marker O4 (O4) markers were studied at 7 and 14 days after stroke.

Six serial coronal sections (10 μm thick) per animal were collected at six regions: bregma + 0.06, − 0.78, − 1.2, − 1.62, − 2.04, − 2.46 mm. Frozen brain sections (10 μm) were fixed in 4% paraformaldehyde for 15 min and washed in 0.01 M PBS (3 × 10 min). Sections were then blocked with 10% goat serum (Sigma) for 60 min to block non-specific binding of the secondary antibody. Sections were then incubated overnight at 4 °C with either rabbit anti-CD3 (1:200; Abcam) or rabbit anti-CD206 (1:500; Abcam). On the following day, they were washed (PBS; 3 × 10 min) and incubated for a maximum of 2 h with either goat anti-rabbit Alexa Fluor 594 (1:500; Thermofisher Scientific) or goat anti-rabbit Alexa Fluor 488 (1:500; Thermofisher Scientific). Finally, sections were again washed and then mounted with Vectashield medium containing 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories), and a coverslip was applied. All tissue-mounted slides were viewed, analyzed and photographed with an Olympus fluorescence microscope. Numbers of immunoreactive cells were counted manually per whole ischemic hemisphere and then averaged across the six regions, as indicated above.

Animals were treated with a probiotic blend consisting of: B. licheniformis, B. indicus HU36™, B. subtilis HU58™, B. clausii, B. coagulans (MegaSporeBiotic™, Microbiome Labs, Saint Augustine, FL, USA), clindamycin (Fresenius Kabi, Lake Zurich, IL, USA), and vancomycin hydrochloride (Hospira, Inc., Lake Forest, IL, USA). Treatments were added onto mouse treats (Bacon Yummies Bio-Serv, Flemington, NJ, USA). Throughout the trial animals were fed sterile Teklad Global 18% Protein extruded rodent diet (Harlan Teklad, Madison, WI, USA). All quantitative real-time polymerase chain reaction (qRT-PCR) primers were obtained from Integrated DNA Technologies (Coralville, IA, USA). Intestinal sections were stained with rabbit anti-CD3 (Abcam, Cambridge, MA, USA), anti-rabbit Alexa 568 (Invitrogen, Eugene, OR, USA), and Anti-Dapi (Littleton, CO, USA).

Tumour tissues were dissected, embedded in tissue freezing medium, and frozen sectioned using freezing microtome (Lecia CM1900, Germany). 10 μm sections fixed in acetone were rinsed with PBS, and were blocked with 2% BSA followed by incubation with rabbit-anti-CD3 (Abcam, 1:100 diluted) at 4 °C overnight. Then sections were incubated with anti-rabbit secondary antibody (Invitrogen, 1:200 diluted) for 1 h at 37 °C. Nuclei were stained with 1 μg/ml DAPI (Sigma). The images were captured using a confocal fluorescence microscope (Olympus) and were analyzed by using FV10-ASW software.