Cy3 conjugated anti rabbit igg

The tissue sections were deparaffinised in xylene, rehydrated in graded ethanol, and boiled in citrate buffer for antigen retrieval. Then, the sections were blocked with 5% BSA for 30 min and incubated with antibodies against CD11c antibody (Thermo Fisher Scientific, Waltham, United States) and IL-6 (Thermo Fisher Scientific, United States) at 4°C overnight. Subsequently, the tissue sections were washed three times with phosphate-buffered saline (PBS) and incubated with secondary antibodies with Alexa Fluor 488-conjugated anti-Syrian hamster IgG (Abcam, Cambridge, United States) and Cy3 conjugated anti-rabbit IgG (Abcam, United States). Finally, the sections were washed and stained with 4′,6-diamidino-2-phenylindole (DAPI). Immunofluorescence images were captured using a microscope (Leica DM13000B, Wetzlar, Germany).

The forebrain tissues were collected and sectioned using a Cryostats. The cortex tissue was then fixed using 100% cold methanol for 10 minutes and permeabilized with 0.1% Triton-X in PBS. The slides were then blocked and incubated with primary rabbit anti-CD68 or anti-claudin-5 (1:1000, Abcam, Cambridge, MA) antibody overnight at 4°C. Following three washes with PBS, cells were incubated with secondary Cy3-conjugated anti-rabbit IgG (1:200, Abcam, Cambridge, MA) for an additional 30 min at room temperature. DAPI was added to dye the nucleus for 5 minutes and 50% glycerinum was used to block the medium. Stained cells were photographed under a fluorescence microscope (Olympus, Tokyo, Japan).

Immunofluorescence analysis was performed according to previously established protocols. Cells were seeded into 24-well dishes and fixed by 4% paraformaldehyde 24 h later. Fixed cells were stained with α-SMA and FAP antibodies (mentioned above), followed by incubation with FITC-conjugated anti-mouse IgG and Cy3-conjugated anti-rabbit IgG (Abcam). Representative images were detected by fluorescence microscopy (Leica, German), and data were processed via Photoshop.

For CD19 staining, the cells were washed with PBS through centrifugation. The cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with saponin (Solarbio, China) for 10 min at room temperature. Subsequently, the cells were subjected to a 1-hour blocking step using 5% BSA and then labeled with CD19 (Intracellular Domain) (D4V4B) XP® Rabbit mAb (Cell Signaling Technology, USA) at 4 ℃ overnight. After washed with PBS for three times, cells were incubated with secondary antibody CY3 conjugated anti-rabbit IgG (Abcam, USA) for 1 h and then stained with Alexa Fluor 488 Mouse Anti-Human CD19 (BD bioscience, USA) for 30 min. Finally, mounting medium with DAPI (Abcam, USA) was added for nuclei staining. The images were collected by Leica DMi8 microscopy (Leica, Germany).
To identify the presence of anti-CD19 CAR on the surface of Nalm-6 cells, the cells were treated with 4% paraformaldehyde for 20 min for fixation, followed by a 1-hour incubation in 5% bovine serum albumin (BSA) for blocking. Then, the cells were incubated and stained with Alexa Flour 647-conjugated Goat Anti-Mouse IgG antibody (Jackson ImmunoResearch, USA) for 1 h followed by Alexa Fluor 488 Mouse Anti-Human CD19 for 30 min. Finally, the cells were counterstained with DAPI and used for confocal images on a Leica TCS SP8 Laser Scanning confocal microscope (Leica Microsystems, Germany).

Immunofluorescence was used to detect the expression of CD44, EpCAM, and MKL-1 in the cells. The cells were grown on a coverslip of 10 centimeters and were fixed in a 4% paraformaldehyde solution followed by permeabilization with 0.5% Triton X-100 (Sigma, USA). Block with goat serum in a dark box for 1 day. The cells were incubated with the corresponding primary antibody overnight at 4°C. Cy3-conjugated anti-rabbit IgG (Abcam, USA) and FITC-conjugated anti-mouse IgG (Abcam, USA) were used as secondary antibodies. After incubation, these cells were incubated for two hours with the nuclei stained by DAPI (Thermo, USA). Ultimately, the samples were coverslipped with a fluorescence microscope.

GeCKOv2 human libraries with 2-vector format (lentiGudie-Puro and LentiCas9-BLAST) were obtained from a commercial source (1000000049; Addgene). The cytomegalovirus (CMV) promoter-driven expression vector, pCAGGS, was used to overexpress ZD17 and two ZD17 truncation mutants, and each gene fused with an S-tag (KETAAAKFERQHMDS) or FLAG at the C terminus. The following antibodies were used in this study: ZDHHC17 polyclonal antibody (15465-1-AP; Proteintech), DYKDDDK-Tag(3B9) mouse antibody (M20008; Abmart), anti-dsRNA MAb J2 (J2-1702; Scicons, Hungary), Cy3-conjugated anti-rabbit IgG (ab6939; Abcam), and DyLight488 anti-mouse IgG (ab96879; Abcam). Anti-S-tag mouse monoclonal antibody was made in-house.