Animal experiments were approved by the Animal Experimentation Ethics Committee at Affiliated Yixing Hospital of Jiangsu University. Twelve female BALB/c nude mice at 4–5 weeks old (SLAC Animal, Shanghai, China) were maintained under normal specific pathogen-free conditions throughout the experiments. To establish a xenograft tumor model, 2 × 106 U2OS cells in 0.1 ml PBS were subcutaneously injected into the right flank of the mice. Ten days after cell inoculation, the mice were randomly divided into two groups and administered TMEM119 siRNA or control siRNA containing formulations via intravenous injection twice a week for 3 weeks (n=6 per group). Tumor growth was monitored were measured every three days as previously described.8 (link) At 45 days after inoculation, the mice were euthanized by cervical dislocation, and tumor weight was recorded. For IHC analyses, tumors were formalin-fixed, paraffin-embedded, sectioned and analyzed using Ki67 immunostaining (Abcam) or the TUNEL assay (Roche).
Ki67 immunostaining
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Ki67 is a nuclear protein that is associated with cellular proliferation. It is expressed during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent in resting cells (G0). Ki67 immunostaining is a method used to detect the presence and distribution of Ki67 in cells, and is commonly used as a marker of cell proliferation in research applications.
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6 protocols using ki67 immunostaining
1
Targeting TMEM119 Inhibits Xenograft Tumor Growth
2
Investigating HDAC1 Knockdown in Glioblastoma
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T98G cells infected with pLVTHM-shRNA negative control (NC) or pLVTHM-HDAC1-shRNA were trypsinized, washed and re-suspended in DMEM without FBS. 12 male athymic nude mice (SLAC Laboratory Animal Center, Shanghai, China) were randomly divided into 2 groups (6 mice/group), and 2×106 T98G cells were subcutaneously injected into the right armpit of the mice. At 46 days after injection, the mice were euthanized and the tumors were excised and weighed. The excised tumor tissues were formalin-fixed, paraffin-embedded, sectioned and then analyzed with TUNEL assay (Roche Applied Science, Mannheim, Germany) or Ki67 immunostaining (Abcam, Cambridge, MA, USA).
3
TREM2 RNAi Inhibits U87 Tumor Growth
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A total of 2 × 106 logarithmically growing U87 cells in 0.1 ml PBS were subcutaneously injected into the right flank of 4-week-old male BALB/c nude mice (n = 10) (SLAC laboratory animal Center, Shanghai, China). Ten days after subcutaneous injection, the nude mice were intratumorally injected with control adenovirus (NC) or TREM2 RNAi adenovirus (TREM-RNAi) (Shanghai SBO Medical Biotechnology Company, China) every two days for 24 days. After that, the mice were killed and tumor tissues were excised and weighed. The excised tumor tissues were formalin-fixed, paraffin-embedded, sectioned and then analyzed with Ki67 immunostaining (Abcam, Cambridge, MA, USA) or TUNEL assay (Roche Applied Science, Mannheim, Germany). All animal experiments were approved by the IACUC committee at the Shanghai Jiaotong University.
4
Tumor Growth Inhibition by TRIM65 Knockdown
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A total of 2 × 106 logarithmically growing NCI-H358 cells with siRNA-NC or siRNA-TRIM65 infection in 0.1 ml PBS were subcutaneously injected into the right armpit of 4-week-old male BALB/c nude mice (n = 6) (SLAC laboratory animal Center, Shanghai, China). 45 days after injection, the nude mice were killed and tumor tissues were excised and weighed. The excised tumor tissues were formalin-fixed, paraffin-embedded, sectioned and then analyzed with Ki67 immunostaining (Abcam, Cambridge, MA, USA) or TUNEL assay (Roche Applied Science, Mannheim, Germany). All animal experiments were approved by the IACUC committee at the Northern Jiangsu People's Hospital and Clinical Medical College of Yangzhou University.
5
Histopathological Analysis of VX2 Tumor
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Tumor tissues were harvested, fixed in 4% paraformaldehyde solution, and embedded in paraffin. These tumor tissues were cut into 5-mm sections for the following histopathological staining preparations: (a) hematoxylin-eosin (HE) to confirm the formation of liver VX2 tumor; (b) terminal deoxynucleotidyl transferase biotin-dUPT nick end labeling (TUNEL) to determine tumor cell apoptosis; and (c) Ki-67 immunostaining (Abcam, Cambridge, MA) to assess tumor proliferations among four groups. Positive cells were imaged using a microscope (200× amplification; Olympus, Tokyo, Japan), and counted by Image-pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD).
The systemic toxicity of Doxo in different groups was also evaluated by hematoxylin-eosin (H&E) staining, which was performed on the lung, heart, kidney, and spleen.
The systemic toxicity of Doxo in different groups was also evaluated by hematoxylin-eosin (H&E) staining, which was performed on the lung, heart, kidney, and spleen.
6
Histopathological Evaluation of Rabbit Liver Post-TACE
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All the rabbits were sacrificed 14 days after TACE by intravenous injection of overdose of pentobarbital sodium for histopathological evaluation. The liver was removed and formaldehyde-fixed, and the paraffin-embedded tissue blocks were prepared. All the paraffin-embedded samples were stained with hematoxylin–eosin (H&E), Ki-67 immunostaining (Abcam, Cambridge, MA) and TUNEL assay (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Moreover, the heart, lung, kidney and spleen were also harvested and stained with H&E for histopathological analysis. All the histopathological pictures were finally taken using the microscopy imaging system (Leica DMi8, Germany).
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