Experiments were performed under an approved National Cancer Institute (NCI) Animal Use Agreement. Female six-week-old athymic nude mice were obtained from Charles River Laboratories (NCI-Frederick Animal Production Area, Frederick, MD). 5×105 MDA-MB-231T cells were injected into the lateral tail vein of each mouse [10 (link)]. A total of 10 mice /experimental group was used. At week 9, mice were sacrificed and lungs collected in Bouin’s solution (70% picric acid, 25% Formaldehyde, 5% glacial acetic acid). Surface lung metastatic lesions were counted before paraffin-embedding and sectioning (10μm) the tissues. Hematoxylin and Eosin (H&E) staining was performed to visualize and count the metastatic lesions in each section. To measure proliferation rate in metastasis, lungs were stained with rabbit anti-Ki67 antibody (Vector Laboratories, Burlingame, CA), following manufacturer’s protocol.
Rabbit anti ki67 antibody
Manufactured by Vector Laboratories
The Rabbit anti-Ki67 antibody is a laboratory reagent used for the detection and quantification of the Ki67 protein, a well-established marker of cellular proliferation. This antibody specifically recognizes the Ki67 nuclear antigen, which is expressed during all active phases of the cell cycle (G1, S, G2, and mitosis) but is absent in resting cells (G0).
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3 protocols using rabbit anti ki67 antibody
1
Quantification of Lung Metastasis in Mice
2
Immunohistochemical Characterization of Neural Markers
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Brains were harvested and fixed in 4% PFA (pH7.4) for a day. The brains were then dehydrated for 24 hrs in 25% sucrose solution. All sections for KI67, DCX, c-Fos and CNPase staining were cut to a thickness of 30 μm on a sliding microtome. Sections were mounted on superfrost slides and dried overnight. Subsequently, slides were incubated in 0.01 mol/L citric buffer for 20 min at 90°C, 3% H2O2 for 10 min, rinsed in PBS, and incubated overnight at room temperature in rabbit anti-KI67 antibody (1:4000, Vector Lab), goat anti-DCX antibody (1:250, Santa Cruz), rabbit anti-c-Fos (1:1000, Santa Cruz), or rabbit anti-CNPase (1:1000, Abcam). The next day, a standard IgG ABC kit (Vector Lab) procedure was used and the slides reacted for 5–10 min with a Sigma DAB tablet. Sections were then counterstained with cresyl violet and cover-slipped with DPX.
For BrdU staining, following a 3% H2O2 incubation for 10 min, slides were subsequently incubated in 2M HCL for 30 min at 37°C. Rat anti-BrdU antibody (1:250, Accurate) was applied overnight. The next day the ABC kit procedure was followed and the slides were reacted with a Sigma DAB tablet. Hippocampus cells were counted bilaterally on every eighth section through the entire rostrocaudal extent of the granule cell layer.
For BrdU staining, following a 3% H2O2 incubation for 10 min, slides were subsequently incubated in 2M HCL for 30 min at 37°C. Rat anti-BrdU antibody (1:250, Accurate) was applied overnight. The next day the ABC kit procedure was followed and the slides were reacted with a Sigma DAB tablet. Hippocampus cells were counted bilaterally on every eighth section through the entire rostrocaudal extent of the granule cell layer.
3
Quantifying Crescents and Proliferation in Kidney Samples
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