CHO cells were fixed with a buffer consisting of 4% formaldehyde (BioLegend) for 15 min at room temperature. Cells were then permeabilized with 0.5% Triton X-100 (BioLegend) for 15 min and treated with a blocking buffer consisting of 2% BSA in PBS for 4 h. To quantify distribution of RNA Poly II, we used an anti-RNA Poly II primary antibody. Detection of RNA Poly II was performed using Alexa-conjugated secondary antibodies (Abcam, Cat. No.150074). Cells were incubated with primary antibodies (diluted with 2% BSA buffer at a ratio of 1:100) overnight at 4 °C. After being washed to remove unbound primary antibodies, cells were incubated with the secondary antibody (diluted with 2% BSA buffer at a ratio of 1:1000) for 1 h under dark conditions. To quantify Poly II fluorescence, the images from the microscope were saved in the Tif format. Image J was used to invert the black and white color. The optical density of the inverted image was calibrated and set to scale. Thresholds were adjusted to get rid of auto-fluorescence background noise and fluorescence intensity in the area of interests was quantified.
Alexa conjugated secondary antibody
Manufactured by Abcam
Sourced in United Kingdom
Alexa-conjugated secondary antibodies are fluorescent-labeled antibodies that bind to the primary antibody, allowing for the detection and visualization of target proteins in various applications, such as immunofluorescence and Western blotting. These antibodies are conjugated with Alexa Fluor dyes, which provide bright and photostable fluorescence signals.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by BioLegend (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Anti p γh2ax, by Cell Signaling Technology (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Anti h3p, by Merck Group (1 mentions)
2 4 amidinophenyl 6 indolecarbamidine dihydrochloride dapi, by Merck Group (1 mentions)
Inverted fluorescence microscope, by Nikon (1 mentions)
6 protocols using alexa conjugated secondary antibody
1
Quantifying RNA Polymerase II Distribution
2
Immunofluorescence Staining of BUMPT Cells
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BUMPT cells were collected and washed once with phosphate-buffered saline (PBS), followed by fixation with 4% paraformaldehyde. After additional washing with PBS, fixed cells were sequentially underwent permeabilization with 0.1% Triton X-100, blocking with 5% bovine serum albumin hydration, and incubation with primary antibodies overnight at 4°C. And then cells were exposed to Alexa-conjugated secondary antibodies(Abcam, Cambridge, MA). Eventually, the sample were examined under fluorescence microscopy.
3
Quantifying RNA Polymerase II Distribution
4
Immunofluorescence Assay for γH2AX
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Cells were seeded in 4-well plates and treated as indicated in each experiment. After 24 h, cells were fixed with methanol at −20 °C for 10 min, and then washed three times using phosphate-buffered saline (PBS). The cells were permeabilized using 0.1% Triton X-100 (Sigma-Aldrich Chemical Co.) and blocked with 5% bovine serum albumin (BSA, Sigma-Aldrich Chemical Co.) in PBS, for 1 h. The cells were incubated with anti-p-γH2AX (Cell Signaling Technology, Beverly, MA, USA) antibody in 5% BSA overnight at 4 °C, and then washed with Tris-buffered saline and Tween 20 (TBST). Following washing for 1 h with anti-rabbit Alexa-conjugated secondary antibody (Abcam, Cambridge, UK) and staining with DAPI, they were observed by fluorescence microscopy.
5
Whole-mount In Situ Hybridization and TUNEL Assay
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Whole-mount colorimetric and fluorescent in situ hybridizations were performed using a detailed protocol as previously described (King and Newmark, 2013 (link); Pearson et al., 2009 (link)). Fluorescence-labeled animals were mounted in ScaleA2 solution (Hama et al., 2011 (link)). Immunostaining with anti-H3P (1:1000, Millipore, Billerica, MA) was performed following fluorescent in situ development and was detected using Alexa-conjugated secondary antibody (1:1000, Abcam, Cambridge, MA). Animals were fixed and stained for TUNEL using a method previously described (Pellettieri et al., 2010 (link)) with modifications: animals were bleached in 0.075% ammonia and 3% hydrogen peroxide and treated with ProteinaseK (2 μg/ml) in PBSTx (0.3% Triton) for 10 min followed by 4% formaldehyde incubation for 10 min prior to TdT reaction.
6
Immunofluorescence Staining of Cellular Markers
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Transfected cells were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.4% Triton X-100 for 10 min. Cells were incubated in 5% normal goat serum for 60 min at room temperature. The serum was drained off, and the slides were blot-dried. Coverslips were incubated with primary antibodies against STEAP3 (Abcam, UK; dilution 1: 100), TfR (Abcam, UK; dilution 1: 100), Nestin (Santa Cruz, USA; dilution 1: 200), and SOX2 (Abcam, UK; dilution 1: 100) at 4 °C overnight followed by incubation with Alexa-conjugated secondary antibody (Abcam, UK; dilution 1: 800) for 1 h. Nuclei were counterstained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Sigma-Aldrich, USA; working dilution 1: 1000) for 5 min at room temperature and mounted in 50% glycerin mounting solution. Rinses in between steps were performed with phosphate buffered saline (PBS). Representative images of STEAP3 and TfR were acquired in confocal microscopy (LSM780, Zeiss). Representative images of Nestin and SOX2 were viewed with a Nikon inverted fluorescence microscope.
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