Cells were grown on cover slides in six-well culture flasks. Prior to fixation, the cells were rinsed in PBS and then fixed for 10 min with cold methanol and 1 min with cold acetone. Thereafter, coverslips were blocked in PBS containing 1% albumin for 1 h at room temperature before being processed for immunofluorescence. The following primary antibodies were used: anti-BUB1 (MAB3610, Millipore, 1 : 10), anti-Centromere FITC-conjugate (15-235-F, Antibodies Incorporated, Davis, CA, USA, 1 : 100), anti-CENP-E (sc-22790, Santa Cruz, 1 : 50), and anti-SURVIVIN (ab469, Abcam, Cambridge, MA, USA, 1 : 100). The following secondary antibodies were used: anti-mouse Cy3-conjugate (AP124C, Millipore, Temecula, CA, USA, 1 : 200), anti-rabbit Cy3 conjugate (AP132C, Millipore, Temecula, CA, USA, 1 : 200). DNA was counterstained with DAPI. Images were acquired on an AxioImager A1 upright microscope (Carl Ziess, Gottingen, Germany).
Ap124c
Manufactured by Merck Group
Sourced in United States
The AP124C is a laboratory equipment product. It is designed for general laboratory applications. The core function of the AP124C is to facilitate various laboratory processes and procedures.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using ap124c
1
Immunofluorescence Analysis of Mitotic Regulators
2
Structured Illumination Microscopy for nSB Detection
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Structured illumination microscopy was performed as described previously [15] . Briefly, a DIG-labeled HSATIII ASO was used to detect nSBs. The DIG-labeled probes were detected using an anti-DIG monoclonal antibody (Abcam, 21H8, ab420, 1:100 dilution) and a
Cy3-conjugated anti-mouse secondary antibody (Millipore, AP124C, 1:100 dilution).
SAFB-positive and HNRNPM-positive cells were visualized using anti-SAFB and anti-HNRNPM antibodies, respectively, and a Cy2-conjugated secondary antibody (Abcam, ab6940, 1:100 dilution). Images were captured using a ELYRA PS.1 microscope (Zeiss) with a 100× objective lens, as described previously [15] .
Cy3-conjugated anti-mouse secondary antibody (Millipore, AP124C, 1:100 dilution).
SAFB-positive and HNRNPM-positive cells were visualized using anti-SAFB and anti-HNRNPM antibodies, respectively, and a Cy2-conjugated secondary antibody (Abcam, ab6940, 1:100 dilution). Images were captured using a ELYRA PS.1 microscope (Zeiss) with a 100× objective lens, as described previously [15] .
3
Visualization of NEAT1 RNA in Cells
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SIM observation was performed as described [19] . Briefly, digoxigenin (DIG)-labeled NEAT1 probes to the 5' end of NEAT1 (+1 to +1000) and the 3' end of NEAT1_2 (+21743 to +22580)
were synthesized using T7 or SP6 RNA polymerase and a DIG RNA labeling kit (Roche) as described [19] . The synthesized probes for NEAT1 5' and 3' ends were used as mixtures to detect shells of paraspeckles. The DIG-labeled probes were detected by anti-DIG monoclonal antibody (Abcam, 21H8, ab420, 1:100 dilution) and anti-mouse secondary antibody conjugated with Cy3 (Millipore, AP124C, 1:100 dilution). GFP-positive cells were visualized by anti-GFP rabbit polyclonal antibody (MBL, 1:100 dilution) and anti-rabbit secondary antibody conjugated with Cy2 (Abcam, ab6940, 1:100 dilution). SIM images were captured using ELYRA PS.1
(ZEISS) with 100× objective lens as described [19] .
were synthesized using T7 or SP6 RNA polymerase and a DIG RNA labeling kit (Roche) as described [19] . The synthesized probes for NEAT1 5' and 3' ends were used as mixtures to detect shells of paraspeckles. The DIG-labeled probes were detected by anti-DIG monoclonal antibody (Abcam, 21H8, ab420, 1:100 dilution) and anti-mouse secondary antibody conjugated with Cy3 (Millipore, AP124C, 1:100 dilution). GFP-positive cells were visualized by anti-GFP rabbit polyclonal antibody (MBL, 1:100 dilution) and anti-rabbit secondary antibody conjugated with Cy2 (Abcam, ab6940, 1:100 dilution). SIM images were captured using ELYRA PS.1
(ZEISS) with 100× objective lens as described [19] .
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