Yeast chromosome spreads technique was performed as described in (56 (link)) with minor modifications. Briefly, yeast strains were grown in log phase in YPG and then shifted for four hours in YPD to deplete Sen1. Cells were converted to spheroplasts and spread on glass slides in presence of SDS 20%. Slides were incubated with 0.25 μg/ml of S9.6 antibody diluted in 80 μl of blocking buffer, washed for 15 min in PBS 1X and then incubated with the secondary Alexa Fluor 488 green antibody (ThermoFisher) diluted 1:1000 in blocking buffer. In vitro RNase H treatment was achieved by incubating the slides, prior to antibody incubations, for 30 min with 2 U of enzyme (NEB) diluted in 80 μl of blocking buffer. Indirect immunofluorescence (IF) was performed using a Zeiss AxioImager M2 microscope with a 40×/NA 1.3 objective.
Alexa fluor 488 green antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 green antibody is a fluorescent dye-labeled antibody used in various biological applications. It is designed to emit green fluorescence when excited by a suitable light source, allowing for the detection and visualization of target molecules in samples.
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Lab products found in correlation
2 protocols using alexa fluor 488 green antibody
1
Yeast Chromosome Spread Technique for R-loop Analysis
2
Immunocytochemical Analysis of CCN2 Expression
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Rat NP cells were plated in 96-well flat-bottom plates (3 × 103 cells/well) and incubated for 24 h. The cells were treated with 1.0 μM BIO, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 (vol:vol) in phosphate-buffered saline (PBS) for 10 min, blocked with PBS containing 10% FBS, and incubated overnight at 4 °C with antibodies against CCN2 (1:100, Santa Cruz Biotechnology). The cells were washed and incubated with an anti-rabbit Alexa Fluor 488 (green) antibody (Thermo Scientific, IN, USA) at 1:200 and with 10 μM 4′,6-diamidino-2-phenylindole (DAPI) for 1 h at room temperature for nuclear staining. The samples were observed under a fluorescence microscope interfaced with a digital imaging system. Cells treated with normal IgG (Cell Signaling Technology) at equal protein concentrations were used as negative controls.
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