For Topoisomerase I-DNA covalent complexes (TOP1cc) immunodetection, cells were fixed with 4% paraformaldehyde in PBS for 15 min on ice, followed by permeabilization in 0.25% TritonX-100 for 15 min at 4 °C. Subsequently, cells were incubated in 1% SDS at room temperature for 5 min followed by five washes in buffer containing 0.1% BSA and 0.1% TritonX-100 in PBS. Coverslips were incubated in blocking buffer containing 10% milk, 150 mM NaCl, and 10 mM Tris-HCl pH 7.4 for 1 h, followed by the addition of anti-Topoisomerase I-DNA Covalent Complexes Antibody, clone 1.1 A (1:250, Millipore-Sigma) and anti-Geminin (1:500, Proteintech) in 5% goat serum in PBS at 4 °C overnight. Cells were rinsed with washing buffer five more times. Secondary antibodies Alexa Fluor 568 goat anti-rabbit (1:1000, Thermo Fisher) and Alexa Fluor 488 goat anti-mouse (1:1000, Thermo Fisher were diluted in 5% goat serum in PBS and applied for 1 h. Cells were rewashed five times in washing buffer, nuclei were stained for 10 min with 1 μg/ml DAPI prior to mounting onto slides with ProLong® Gold Antifade Mountant (Invitrogen life technology).
Anti geminin
Manufactured by Proteintech
Anti-Geminin is a primary antibody that targets the Geminin protein, which is involved in the regulation of DNA replication during the cell cycle. This antibody can be used for the detection and analysis of Geminin expression in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Blocking solution, by Agilent Technologies (1 mentions)
Anti rabbit cy5, by Thermo Fisher Scientific (1 mentions)
Ventana discovery xt, by Roche (1 mentions)
2 protocols using anti geminin
1
Immunodetection of Topoisomerase I-DNA Complexes
2
Immunofluorescence Staining of Geminin in Tissue Sections
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Tumors were harvested, formalin-fixed, and paraffin-embedded. According to the manufacturer’s instructions, 4-μm-thick tissue sections were cut with a microtome and stained using the automated Ventana Discovery XT staining system (Ventana Medical Systems). Antigen retrieval was performed in Cell Conditioning 1 solution, and slides were incubated with anti-Geminin (Proteintech Group) antibodies in PBS at 37°C for 60 minutes. A negative control slide using PBS instead of the primary antibody was prepared in parallel. On the bench, slides were incubated for 20 minutes with blocking solution (Dako, Agilent Technologies) followed by washing with PBS and by incubation with the secondary antibody (anti-rabbit Cy5; Life Technologies Inc.) in PBS at room temperature for 45 minutes. Finally, after washing with PBS, slides were incubated for 15 minutes at room temperature with a 0.1% (w/v) solution of Sudan Black in 70% ethanol to quench tissue autofluorescence. Following a PBS wash, slides were mounted using ProLong Gold Antifade Mountant with DAPI and stored at 4°C. Images were taken with a Zeiss microscope and quantification was performed with ImageJ software.
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