EPC, RTG2 and RTgill cells were seeded onto Poly-L-Lysine (0.01% w/v) coated coverslips (Sigma-Aldrich, St. Louis, MO, USA). Following indicated treatments, cells were fixed with 4% paraformaldehyde (BM-155; Boston Bioproducts, Ashland, MA, USA) for 15 min at room temperature and then permeabilized with 0.2% Triton X-100 in PBS for 15 min at room temperature. Cells were then blocked with 3% BSA, 0.02% Tween in PBS for 1 h at room temperature and then incubated the indicated antibodies at a 1:300 dilution in 3% BSA in PBS overnight at 4 °C. Cells were then incubated with a secondary antibody at a 1:300 dilution in PBS (Alexa 488- or Alexa 647-conjugated anti-immunoglobulin antibody, Molecular Probes; Eugene, OR, USA) for 1 h at room temperature. Cell nuclei were stained with DAPI (17985–50; EMS, Hatfield, PA, USA). Cells were imaged on an Olympus IX81 inverted fluorescence microscope and the analysis and processing of images were performed using Stream View and ImageJ software [70 (link)]. Cells containing G3BP1 puncta (n > 5) were considered stress granule positive and counted. The percentage of cells forming stress granules was calculated from at least 100 cells from various fields. Data are representative of three independent experiments.
Alexa 488 or alexa 647 conjugated anti immunoglobulin antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa 488- or Alexa 647-conjugated anti-immunoglobulin antibody is a laboratory reagent used for the detection and quantification of immunoglobulin (antibody) molecules in various biological samples. The antibody is conjugated with either Alexa Fluor 488 or Alexa Fluor 647 dyes, which serve as fluorescent labels that can be detected using appropriate instrumentation.
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Lab products found in correlation
Ix81 inverted fluorescence microscope, by Olympus (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Bm 155, by Boston BioProducts (1 mentions)
Las af software, by Leica (1 mentions)
Paraformaldehyde (pfa), by Boston BioProducts (1 mentions)
Vectashield with dapi to stain the nucleus, by Vector Laboratories (1 mentions)
2 protocols using alexa 488 or alexa 647 conjugated anti immunoglobulin antibody
1
Stress Granule Formation Assay
2
Immunofluorescence Analysis of Stress Granules
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Cells were cultured on glass coverslips and after treatment, cells were fixed with 4% paraformaldehyde (Boston Bioproducts) for 15 minutes and permeabilized with 0.1% Triton X-100 in PBS for 15 minutes. Cells were then blocked with 3% BSA for 1 hour at room temperature and incubated overnight at 4°C with indicated antibodies. Alexa488-or Alexa647-conjugated anti-immunoglobulin antibody (Molecular Probes) were used as secondary antibodies. Cell nuclei were stained with Vectashield with DAPI to stain the nucleus (Vector Labs). Fluorescence and confocal microscopy assessments were performed with Leica CS SP5 multi-photon laser scanning confocal microscope (Leica Microsystems). All subsequent analysis and processing of images were performed using the LAS AF software (Leica Microsystems). Cells containing avSG (n>5) which are above 0.6µm in diameter were considered for analysis. The percentage of avSG containing cells were calculated in at least five random fields from a minimum of 100 cells per treatment.
Colocalization of proteins in stress granules were assessed by line scan analysis using Image J as described (74) (link). A line was drawn across the stress granules and the intensity were measured using plot profile. The arbitrary intensity was plotted according to arbitrary distance for each channel.
Colocalization of proteins in stress granules were assessed by line scan analysis using Image J as described (74) (link). A line was drawn across the stress granules and the intensity were measured using plot profile. The arbitrary intensity was plotted according to arbitrary distance for each channel.
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