Ab150084

All the transplanted tumor samples were fixed by 4% PFA at 4 °C overnight and embedded into paraffin. Paraffin-embedded tissues were sectioned into 5 μm sections. The sections were deparaffinized and rehydrated by dimethylbenzene, gradient ethanol series and double-distilled water. After washed by PBS three times for 5 min, antigen retrieval was performed by boiling the sections in citric acid buffer (PH6.0) for 15 min. Cooled sections were washed by PBS, blocked by 5% normal goat serum for 45 min, and incubated with rabbit anti-human CD8 antibody at dilution of 1:100 overnight at 4 °C. Next day, the sections were stained by Alexa Fluor 594-conjugated goat anti-Rabbit IgG antibody (ab150084, Abcam), followed by DAPI staining. Slides were observed under Zeiss Observer A1 microscope. The mean number of CD8+ T cells in four microscopic fields of 40x objective was scored independently by two authors in a blinded manner.

Immunofluorescence staining was performed to determine the composition of microtissues. The microtissues were fixed with 4% paraformaldehyde, and then dehydrated with 20% and 30% sucrose, respectively. Before staining, the microtissues were cut into 9-μm flakes using a frozen slicer. Subsequently, the samples were blocked with 10% goat serum (SL038; Solarbio) for 1 h at room temperature after washing with PBS. Then, the samples were incubated overnight at 4 °C with Mouse anti-laminin antibody (1:200; ab 242198; Abcam) and Rabbit anti-fibronectin antibody (1:500; ab268020; Abcam). Next, they were incubated with Goat Anti-Mouse IgG H&L (Alexa Fluor® 488; 1:200; ab150117; Abcam) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594; 1:200; ab150084; Abcam) secondary antibodies for 2 h at room temperature. 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) was used for 10 min at room temperature to stain the nuclei. Images were taken using a fluorescence microscope (PANNORAMIC Confocal; 3DHISTECH).

Representative animals were selected for analysis. All the rats used in the experiment were initially inoculated subcutaneously with an amount of 50,000 NS1 rat glioma cells. Tumors isolated from the rats were fixed using Phosphate-buffered 4% Paraformaldehyde as described previously [3 (link)], paraffin embedded and sectioned using a microtome. The primary polyclonal antibodies were diluted 1:400 in PBS containing 1% BSA and 2% normal goat serum in the following manner; the fixed tissue samples were incubated with rabbit anti-rat C1 inactivator (Covance, USA), permeabilized with 0.1% Triton^™ X-100 for 10 minutes, blocked with 1% BSA for 1 hour and labeled with 2 μg/mL the rat primary antibody for 1 hours at room temperature. The tissue was subsequently washed and incubated for 1 hour at room temperature with secondary antibodies consisting of Alexa Fluor 594-conjugated goat anti-rabbit serum (ab150084 Abcam) at a concentration of 0.5μg/mL in phosphate buffered saline containing 0.2% BSA at room temperature. After washing with PBS, the tissue was mounted with anti-fading vecta-shield mounting medium with 4,6-diamidino-2-phenylindole (nuclear stain with DAPI) (Vector Laboratories Inc., Burlingame, USA) and were photographed using fluorescence microscope fitted with the appropriate wavelength filters.

Adipose-derived stem cells were seeded on coverslips covered with poly-L-lysine (No. P8920; Sigma) in 6-well plates and the immunofluorescence method was applied to show SC associated biomarkers.
After incubation for 24 h, the medium in the wells was removed. Coverslips with attached cells were then washed with 1 × PBS. The cells were then fixed with 4% paraformaldehyde for 10 min. After washing three times with PBS for 5 min each time, ADSCs were blocked with 10% goat serum (SL038; Solarbio) in PBS for 1 h. Primary antibody anti-S100β (1:200, ab52642, Abcam, United States); It was dropped onto coverslips and incubated at room temperature overnight. Next, secondary antibody, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594; 1:200; ab150084; Abcam), was dropped onto coverslips. After incubation with secondary antibodies in the dark for 1.5 h, the coverslips were washed and incubated with DAPI (No. H-1200; Vector Laboratories Inc., Burlingame, CA, United States). For the final step, the coverslips were inverted onto the slides and analyzed under a fluorescent microscope. The number of surviving cells, and the rates of S100β - positive cells in the 5 groups of ADSCs were calculated according to 5 randomly selected fields in each group at 200 × magnification using IPP 6.0.

HTR-8/SVneo cells (4 × 10⁵) were treated with circ-CCNB1 vector or control vector for 48 hours, incubated with 10 µM 5-bromo-2-deoxyuridine (BrdU) (Beyotime, Shanghai, China) for 30 minutes at 37 ºC in the dark, and then washed with phosphate-buffered saline (PBS) and fixed in cold 70% ethanol for 30 minutes. The fixed cells were treated with 1 M HCl and blocked with 1% BSA (Beyotime) at room temperature for 1 hour, followed by a monoclonal antibody against BrdU (1:200; #ab152095, Abcam, RRID: AB_2813902) for 1 hours and Alexa Fluor 594 secondary antibody (1:1000; #ab150084 Abcam, RRID: AB_2734147) for 30 minutes at room temperature. DAPI (Beyotime) was used to stain the nuclei. Images were visualized in 3 randomly selected areas with 3 experimental replicates using an inverted fluorescence microscope (Leica, Wetzlar, Germany).

Cells were fixed in 4% paraformaldehyde for 15 min, followed by three washes of DPBS, blocked and permeabilized in PBS containing 0.1–0.2% Triton X-100 and 10% horse serum. The coverslips were incubated with primary antibodies; for NPCs: rabbit anti-PAX6 (CST, mAb#60433, 1:250) and mouse anti-NESTIN (CST, mAb#33475,1:2000); for Neurons: chicken anti-MAP2 (Abcam, ab92434, 1:500), rabbit anti-TBR1 (Abcam, ab183032, 1:250), rabbit anti-VGLuT1 (Abcam, ab227805, 1:500) and Mouse anti-GABA (Abcam, ab86186, 1:400) in the blocking solution overnight at 4 °C. On the next day, they were washed in DPBS and incubated with DAPI (Abcam, ab228549, 1:1000) and corresponding secondary antibodies (Abcam, ab150084, ab150117, ab175711, 1:250) for 60 min at room temperature. Then the coverslips were washed three times, mounted on glass slides using Fluromount-G (mounting medium), and dried overnight while being protected from light. Fluorescence signals were detected using a Leica THUNDER imager and analyzed using ImageJ and MATLAB.