Alexa fluor 594

The adult Cx3cr1-GFP mouse brain slices (50-μm-thick) were incubated with blocking solution (0.01 M PBS/10% normal donkey serum/0.1% Triton X-100) for 1 hr at room temperature. The slices were transferred to primary antibody (anti-GFP, Aves, GFP-1020, 1:250) dilutions for overnight at 4 °C followed by washing with PBST (0.1% Triton X-100 in PBS) for three times (5 min/each time), then to secondary antibody dilutions (donkey anti-chicken Alexa Fluor 594, Jackson Immunoresearch, 117978, 1:500) for 2 hr at room temperature, then washed in PBS for several times.
The whole embryos (E12.5) was immunostained for neurofilament based on iDISCO protocol with methanol pretreatment^{34 (link)}. The pretreated sample was incubated in PBS/0.2% Triton X-100/20% DMSO/0.3 M glycine overnight at 37 °C, blocked in PBS/0.2% Triton X-100/10%DMSO/6% Goat Serum for 1d at 37 °C. After transferring to primary antibody dilutions (anti NF-200, DHSB, 2H3, 1:100) for 3d followed by washing, and then incubated in secondary antibody dilutions (goat anti-mouse Alexa Fluor 594, Jackson Immunoresearch, 115–585–146, 1:300) for 2d at room temperature and finally washed for 1d prior to clearing and imaging.

VVEC were plated on eight-well chamber slides (Labtek, Grand Rapids, MI), serum starved, and stimulated with ATP (100 μM) for 0.5–24 h. Stimulated cells were washed three times with PBS (5 min each) and blocked in PBS containing 5% normal goat serum and 0.3% Triton X for 1 h at room temperature. Cells were incubated with rabbit polyclonal antibody against GLUT-1 (Abcam no. 115730; 1:400) overnight at 4°C. After the incubation, cells were washed as indicated above and incubated with fluorescein-conjugated Grifonia Simplicifolia lectin (Vector Labs, Burlingame, CA; no. FL-1101) at the concentration of 2 μg/ml for 1 h at room temperature. Following this step, cells were washed and incubated with the secondary, goat anti-rabbit antibody Alexa Fluor-594 (Jackson Labs, Farmington, CT; no. 111-585-003; 1:400), for 1 h at room temperature. Finally, cells were washed, air dry in the dark, and treated with Prolong Gold anti-fade with 4,6-diamidino-2-phenylindole (Cell Signaling Technology, no. 8961-I). Images were captured using EVOS imaging system under ×40 magnification.

Mouse liver tissues were fixed in 4% paraformaldehyde overnight at 4 °C and then embedded in OCT (Sakura). Five micrometer Frozen sections were prepared using a Cryotome FSE cryostat (Thermo-Fisher Scientific). The tissue sections were incubated in the blocking buffer (5% donkey serum, 0.3% Triton-X 100 in PBS) at room temperature for 1 h followed by the staining with primary antibodies. The following primary antibodies were used: Rat anti-mouse F4/80 (clone CI:A3-1, ab6640, dilution 1/500, Abcam) and Rabbit anti-mouse phosphor-AMPKα (Thr172) antibody (clone 40H9, #2535, dilution 1/200, Cell Signaling Technology). Then slides were washed and incubated for 1 h with the following secondary antibodies: donkey anti-rat Alexa Fluor 488 and donkey anti-rabbit Alexa Fluor 594 (Jackson ImmunoResearch Laboratories). Sections were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) before being mounted. All immunofluorescence staining was performed in the dark. Imaging was performed using a Zeiss LSM 880 and images were processed using Zeiss ZEN software.

Murine anti-CD19 CAR T cells were generated from T cells collected from the spleens of balb/c mice by transducing the T cells with a retroviral vector (MSGV1-1D3-28Z.1-3 mut) kindly provided by James Kochenderfer & Steven Rosenberg (Addgene plasmid # 107227; http://n2t.net/addgene:107227; RRID : Addgene_107227) (25 (link)). Splenic T cells were then isolated by negative selection (EasySep™ Cat# 19851) and activated with anti-CD3/CD28-conjugated Dynabeads (Gibco™ Cat# 11453D) for 24 hours prior to their transfer to RetroNectin-coated plates for transduction with the above vector. To boost transfection efficiency, second transduction was performed one day after the first, and the cells were cultured for an additional 3 days. Transduction efficiency was quantitated using an anti-rat IgG F(ab’)₂ fragment conjugated to Alexa Fluor 594 (Jackson ImmunoResearch Laboratories Cat# 212-586-168).