Slides with freshly made metaphase spreads were put through an ethanol dehydration series and air dried. Cells and specific centromere probe (LPE 001R or LPE 009R; Cytocell) were denatured for 2 min at 75°C then incubated overnight at 37°C. The following day, slides were washed with 0.25X SSC at 72°C followed by a brief wash in 2X SSC, 0.05% Tween. DNA was stained for 6 min with 0.5 μg/ml DAPI (Roche) and coverslips mounted in Vectashield (Vector H‐1000, Vector Laboratories).
Vectashield vector h 1000
Manufactured by Vector Laboratories
Sourced in United Kingdom
Vectashield (Vector H-1000) is a mounting medium designed for use in fluorescence microscopy. It is a water-based, non-hardening solution that helps to preserve the fluorescence of tagged molecules in fixed samples. Vectashield is intended to be used as a mounting medium to facilitate imaging of fluorescently labeled specimens.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Roche (5 mentions)
A11012, by Thermo Fisher Scientific (4 mentions)
A11017, by Thermo Fisher Scientific (2 mentions)
A21445, by Thermo Fisher Scientific (2 mentions)
A11008, by Thermo Fisher Scientific (2 mentions)
α tubulin, by Abcam (1 mentions)
Cenp a, by Abcam (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Rpa70, by Abcam (1 mentions)
γh2ax, by Merck Group (1 mentions)
Ab5093, by Abcam (1 mentions)
Ab6046, by Abcam (1 mentions)
Anti mouse af 647, by Jackson ImmunoResearch (1 mentions)
Ab97777, by Abcam (1 mentions)
5 protocols using vectashield vector h 1000
1
Fluorescence In Situ Hybridization Protocol
2
Immunofluorescence Staining of Cellular Components
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Cells grown on coverslips were fixed in freshly-prepared PTEMF (0.2% Triton X-100, 0.02 M PIPES (pH 6.8), 0.01 M EGTA, 1 mM MgCl2, 4% formaldehyde) for 10 min at room temperature. For cold treatment assays cells were placed on ice for 10 min before fixation. After blocking with 3% BSA, cells were incubated with primary antibodies: α-tubulin at 1:600 (Abcam abID#6046, Cambridge, UK), Centrin3 at 1:500 (Abcam abID#54531), CREST at 1:400 (Antibodies Incorporated, #15-234-0001, Davis, CA), CENP-A at 1:400 (Abcam abID#13939), BubR1 at 1:500 (Cambridge Bioscience), Mad2 at 1:500 (Bethyl Lab, A300–300A). Secondary antibodies used were goat anti-mouse AlexaFluor488 (A11017, Invitrogen, UK), goat anti-rabbit AF594, AF488 (A11012, A11008, Invitrogen), and goat anti-human AF647 (109-606-088-JIR, Stratechor A21445, Invitrogen). DNA was stained for 6 min with DAPI (Roche, UK) and coverslips mounted in Vectashield (Vector H-1000, Vector Laboratories, Peterborough, UK).
3
Immunofluorescence Assay for DNA Damage Response
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Cells grown on glass slides or coverslips were fixed with PTEMF (0.2% Triton X-100, 0.02 M PIPES (pH 6.8), 0.01 M EGTA, 1 mM MgCl2, 4% formaldehyde). After blocking with 3% BSA, cells were incubated with primary antibodies according to suppliers’ instructions: CREST (Antibodies Incorporated, 15-234-0001), γH2aX (Millipore, 05-636), RPA70 (Abcam, ab79398). Secondary antibodies used were goat anti-mouse AlexaFluor 488 (A11017, Invitrogen), goat anti-rabbit AF594, AF488 (A11012, A11008, Invitrogen), and goat anti-human AF647 (109-606-088-JIR, Stratech or A21445, Invitrogen). DNA was stained with DAPI (Roche) and coverslips mounted in Vectashield (Vector H-1000, Vector Laboratories). EdU incorporation and staining was achieved using the Click-It kit (Life Technologies), following the manufacturer’s instructions.
4
Immunofluorescence Assay for Cytoskeletal and Centromeric Proteins
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Cells grown on glass slides or coverslips were fixed with PTEMF (0.2% Triton X-100, 0.02 M PIPES [pH 6.8], 0.01 M EGTA, 1 mM MgCl2, and 4% formaldehyde). After blocking with 3% BSA, cells were incubated with primary antibodies according to suppliers’ instructions: beta-tubulin (ab6046; Abcam), Centrin 3 (ab54531; Abcam), CREST (15-234-0001; Antibodies Incorporated), and CENP-E (ab5093; Abcam). Secondary antibodies used were goat anti-mouse Alexa Fluor 488 (A11017; Invitrogen), goat anti-rabbit AF594 and AF488 (A11012 and A11008; Invitrogen), and goat anti-human AF647 (109-606-088-JIR [Stratech] or A21445 [Invitrogen]). DNA was stained with DAPI (Roche), and coverslips were mounted in Vectashield (Vector H-1000; Vector Laboratories).
5
Immunofluorescence Staining of Kinetochore Proteins
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Cells grown on glass slides or coverslips were fixed with PTEMF (0.2% Triton X‐100, 0.02 M PIPES [pH 6.8], 0.01 M EGTA, 1 mM MgCl2, and 4% formaldehyde). After blocking with 3% BSA PBS for 1 h at 21°C, cells were incubated with primary antibodies in blocking buffer for 1 h at 21°C: 1:1,000 α‐tubulin (118M4779V; Sigma Aldrich), 1:250 CREST (15‐234‐0001; Antibodies Incorporated), 1:500 CENP‐T (ab220280; Abcam), 1:500 KNL‐1 (ab222055; Abcam), 1:500 Mad2 (ab97777; Abcam), 1:500 Ndc80 (ma1‐213308; Invitrogen). For Ndc80 staining, blocking was conducted instead in 5% Milk PBS. Secondary antibodies used were goat anti‐human AF647 (109–606‐088‐JIR; Stratech), goat anti‐rabbit AF594 (A11012; Invitrogen), goat anti‐mouse AF594 (A11005; Invitrogen) and anti‐mouse AF647 (115–605‐003‐JIR; Jackson Immunoresearch) at 1:250 dilution. DNA was stained with DAPI (Roche), and coverslips mounted in Vectashield (Vector H‐1000; Vector Laboratories). Following each antibody incubation, cells were washed in PBS three times, each for 3 min.
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