The recombination assays were fixed and permeabilized as described above. Then blocked overnight at 4°C in 10% DS, 1% BSA, 1.8% IgG MOM and 0.1% PBST. Primary antibodies were incubated overnight at 4°C in 16% MOM and 0.1% PBST: Pna-Rhodamine (1:75), rabbit anti-K5 (1:2000), mouse anti-Tubb3 (1:200), rabbit anti-Ecadherin (1:100), rat anti-perlecan (1:100). Primary antibodies for human-mouse recombination incubated overnight at 4°C in 16% MOM and 0.1% PBST: mouse anti-Ki67 (1:100), goat anti-Kit (1:100), mouse anti-human nuclear antigen (1:100, Millipore), rabbit anti-Ecadherin (1:100), mouse anti-Tubb3 (1:200), rat anti-integrinβ1 (1:100, Gift of Dr. K.M. Yamada) and rabbit anti-K5 (1:2000). Secondary antibodies incubated for one hour at room temperature in 16% MOM protein and 0.1% PBST: 1:2000 Hoescht, anti-Rabbit cy3 and cy5 (1:200), anti-Mouse cy2 (1:100), anti-goat cy 5 (1:200), and anti-rat cy3 and cy2 (1:200).
Mouse anti ki67
Manufactured by Merck Group
The Mouse anti-Ki67 is a laboratory equipment used in research and diagnostic applications. It is an antibody that specifically binds to the Ki67 protein, which is a marker for cell proliferation. The Ki67 protein is present during all active phases of the cell cycle, but is absent in resting cells. This makes the Mouse anti-Ki67 a useful tool for identifying and quantifying proliferating cells.
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Lab products found in correlation
Mouse anti human nuclear antigen, by Merck Group (1 mentions)
Hardset mounting media with dapi, by Vector Laboratories (1 mentions)
4 well chamber slide, by Thermo Fisher Scientific (1 mentions)
Mouse anti coronin 1a, by Santa Cruz Biotechnology (1 mentions)
Mouse anti vimentin, by Santa Cruz Biotechnology (1 mentions)
Anti gfap, by Agilent Technologies (1 mentions)
Axio imager z1 microscope, by Zeiss (1 mentions)
Mouse anti human α sma, by Merck Group (1 mentions)
Rat anti f4 80, by Bio-Rad (1 mentions)
Axioskop fluorescence microscope, by Zeiss (1 mentions)
Mouse monoclonal anti pcna, by Merck Group (1 mentions)
Falcon eight well culture slides, by BD (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Mouse anti pcna, by Abcam (1 mentions)
Anti α tubulin 12g10, by Developmental Studies Hybridoma Bank (1 mentions)
5 protocols using mouse anti ki67
1
Immunofluorescence Staining of Recombination Assays
2
Immunofluorescence Analysis of Astrocytes
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Astrocytes were seeded in a 4‐well chamber slide (Nunc, Cat# 177437) at a density of 30,000 cells per well. Cells were fixed after 24 h, using 4% paraformaldehyde, washed thrice with 1X PBS, blocked, and permeabilized using 4% BSA containing 0.4% Triton‐X 100. The cells were then incubated with antibodies, mouse anti‐coronin 1A (Santa Cruz Biotechnology, 1:100, Cat# sc‐100925, RRID:AB_2291951), mouse anti‐Ki 67 (Millipore, 1:1000, Cat# MAB4190, RRID:AB_95092), mouse anti‐vimentin (Santa Cruz Biotechnology, 1:1000, Cat# sc‐6260, RRID:AB_628437), overnight at 4°C, or anti‐GFAP (Dako, 1:1000, Cat# Z0334, RRID:AB_10013382) for 1 h at 25°C. The cells were then washed thrice with 1X PBS and incubated with appropriate fluorophore‐tagged secondary antibodies (Invitrogen, 1:1000). The wells were washed thrice with 1X PBS and mounted using Hardset mounting media with DAPI (Vector Labs, Cat# H‐1500). For each group, a minimum of five images were captured from random fields using the AxioImager Z1 microscope (Zeiss).
3
Immunohistochemical Profiling of Liver Metastases
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Liver sections (7 µm thick) were fixed in cold acetone, and stained with hematoxylin/eosin staining. For triple immunohistochemical analyses, sections were reacted with rat anti-mouse CD31 (1:100, Dako), rabbit anti-mouse Desmin (1:200, Dako), and mouse anti-human α-SMA (1:50, Sigma-Aldrich) followed by the appropriate fluorescent secondary antibodies (n = 72). Serial sections were incubated with either rat anti-F4/80 (1:100, BIO-RAD) (n = 36) or mouse anti-Ki67 (1:200; Sigma-Aldrich) (n = 36), followed by appropriate fluorescent secondary antibodies. Irrelevant appropriate immunoglobulins were used as negative controls. Metastases were microphotographed using a Zeiss Axioskop fluorescence microscope under the same conditions. Specific immunostaining was quantified using FIJI-ImageJ software. Results were expressed as the percentage of specifically colored tissue area relative to the whole micrometastatic foci area.
4
Proliferative Potential of SurR9-C84A in Differentiated SK-N-SH Cells
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To evaluate the proliferative potential of SurR9-C84A on differentiated SK-N-SH cells, immunofluorescence for the proliferative markers PCNA and Ki67 were studied. Cells were seeded at 105 cells/well in BD Falcon eight-well culture slides following the protocol described for the undifferentiated cells. After treatment with SurR9-C84A, the differentiated cells were incubated with mouse monoclonal anti-PCNA and mouse anti-Ki67 (1:100; Sigma-Aldrich) for 1 hour at 37°C. FITC-labeled rabbit antimouse and goat antirabbit (1:100) were used as secondary antibodies. The cell nucleus was stained with PI (1:500) for 30 minutes, and further imaging was carried out using the Leica SP5 confocal microscope at 40× magnification.
5
Immunodetection of Cell Proliferation
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Mouse anti-BrdU (Caltag Laboratories, San Francisco, CA), mouse anti-Ki67 (Sigma), mouse anti-PCNA (Abcam, Cambridge, MA), rabbit anti-phospho-STAT5 (Tyr694), rabbit anti-phospho-Erk1/2 (Thr202/Tyr204; Cell Signaling Technology) antibodies were purchased commercially. Anti-α-tubulin (12G10) was obtained from the Developmental Studies Hybridoma Bank (NICHD and The University of Iowa, Department of Biology Iowa City, IA).
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