For cytology, 1 × 108 cells were harvested at the indicated time point and yeast chromosome spreads were prepared as described in Grubb et al. (2015) (link). Primary antibodies used were mouse monoclonal 9E11 anti-myc antibody (dilution 1:200), rabbit polyclonal anti-Zip1 antibody (dilution 1:100; Santa Cruz Biotechnology sc-33733), rabbit monoclonal anti-Red1 antibody (#16441; dilution 1:200; a gift from N. Hollingsworth), and rabbit anti-Gmc2 antibody (dilution 1:1600; a gift from Amy MacQueen). The secondary antibodies were Alexa488-conjugated goat antirabbit (dilution 1:200; Thermo Fischer Scientific A-11008) and Alexa568-conjugated goat antimouse (dilution 1:200; Thermo Fischer Scientific A-11004). Chromosomal DNA was stained by 4,6-diamidino-2-phenylindole (DAPI). Fluorescence images were visualized and acquired using the Deltavision IX70 system (Applied Precision), 100× objective, and softWoRx imaging software. Images were processed by deconvolution using the constrained iterative deconvolution algorithm within softWoRx. Image analysis and signal quantification were performed using the Fiji software and R scripts. Fluorescence intensity was measured as the average sum of pixel density of Zip1 stretches per nucleus.
Alexa568 conjugated goat antimouse
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa568-conjugated goat antimouse is a fluorescently labeled secondary antibody used for detection in various immunological techniques. It consists of goat-derived antibodies that specifically bind to mouse primary antibodies, with the Alexa568 dye attached to provide a fluorescent signal. This product can be used to visualize the localization and abundance of target antigens recognized by mouse primary antibodies in samples.
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Lab products found in correlation
Alexa 488 conjugated goat anti rabbit, by Thermo Fisher Scientific (4 mentions)
Deltavision ix70 system, by Cytiva (2 mentions)
9e11 anti myc antibody, by Santa Cruz Biotechnology (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Prolong diamond, by Thermo Fisher Scientific (1 mentions)
Anti p53 do 1, by Santa Cruz Biotechnology (1 mentions)
Alexa647 conjugated goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Lsm 780, by Zeiss (1 mentions)
Alexa 568 conjugated goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Mouse anti gfp, by Merck Group (1 mentions)
Alexa 488 conjugated goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Rabbit anti dsred, by Takara Bio (1 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (1 mentions)
5 protocols using alexa568 conjugated goat antimouse
1
Yeast Chromosome Spread Immunofluorescence
2
Immunofluorescence Colocalization Assay
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For the immunofluorescence colocalization assay, 105 cells/wells were seeded in 24-well plates and allowed to adhere overnight. The cells were then washed with PBS, fixed with a methanol/acetone solution (1:1) for 10 min at −20°C, and incubated for 2 h with 100 µL of primary antibodies at 37°C with 5% CO2. The following antibodies were used: anti-p53 DO-1 (Santa Cruz Biotechnology, USA) (1:200) and A11 anti-amyloid oligomers (Millipore) (1:1,000) in blocking buffer (10% glycerol, 0.2% Tween 20, and 2% BSA in PBS). Subsequently, the cells were incubated with Hoechst 33258 (ThermoFisher, USA) (1:1,000), Alexa 568-conjugated goat anti-mouse, and Alexa 647-conjugated goat anti-rabbit (ThermoFisher, USA) secondary antibodies (1:2,000) for 1 h at room temperature, protected from light. After washing with PBS, coverslips were mounted with ProLong Diamond (ThermoFisher, USA) and analyzed by confocal microscopy (Leica TCS SPE confocal microscope, Carl Zeiss Inc.).
3
Cytological Analysis of Meiotic Chromosome Dynamics
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For cytology, 1×10 8 cells were harvested at the indicated time-point and yeast chromosome spreads were prepared as described in (Grubb et al. 2015) . Primary antibodies used were mouse monoclonal 9E11 anti-myc antibody (dilution 1:200), (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted August 13, 2021. ; https://doi.org/10.1101/2021.08.13.456249 doi: bioRxiv preprint rabbit polyclonal anti-Zip1 antibody (sc-33733, SantaCruz Biotech, dilution 1:100) and rabbit monoclonal anti-Red1 antibody (#16441, Gift from N. Hollingsworth, dilution 1:200). The secondary antibodies were Alexa488-conjugated goat anti-rabbit (A-11008, Thermo Fischer Scientific; dilution 1:200), Alexa568-conjugated goat antimouse (A-11004, Thermo Fischer Scientific; dilution 1:200) . Chromosomal DNA was stained by 4,6-diamidino-2-phenylindole (DAPI). Fluorescence images were visualized and acquired using the Deltavision IX70 system (Applied Precision), objective 100X and softWoRx imaging software. Images were processed by deconvolution using the constrained iterative deconvolution algorithm within softWoRx. Image analysis and signal quantification was performed using the Fiji software and R-scripts. Fluorescence intensity was measured as the sum of pixel density of Zip1 stretches.
The copyright holder for this preprint this version posted August 13, 2021. ; https://doi.org/10.1101/2021.08.13.456249 doi: bioRxiv preprint rabbit polyclonal anti-Zip1 antibody (sc-33733, SantaCruz Biotech, dilution 1:100) and rabbit monoclonal anti-Red1 antibody (#16441, Gift from N. Hollingsworth, dilution 1:200). The secondary antibodies were Alexa488-conjugated goat anti-rabbit (A-11008, Thermo Fischer Scientific; dilution 1:200), Alexa568-conjugated goat antimouse (A-11004, Thermo Fischer Scientific; dilution 1:200) . Chromosomal DNA was stained by 4,6-diamidino-2-phenylindole (DAPI). Fluorescence images were visualized and acquired using the Deltavision IX70 system (Applied Precision), objective 100X and softWoRx imaging software. Images were processed by deconvolution using the constrained iterative deconvolution algorithm within softWoRx. Image analysis and signal quantification was performed using the Fiji software and R-scripts. Fluorescence intensity was measured as the sum of pixel density of Zip1 stretches.
4
Immunostaining of Kv2.1, VGAT, and CB1R in Mouse Amygdala
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WT and KO mice were perfused with 4% paraformaldehyde in phosphate-buffered saline and whole brains were fixed overnight at +4 °C. Brains were sectioned at 70 µm and slices containing amygdala were selected for immunostaining. Immunostainings were performed with standard procedures using the following combination of primary and secondary antibodies: Mouse anti-Kv2.1 (Neuromab) and Alexa 568-conjugated goat anti-mouse (Invitrogen); Guinea pig anti-vesicular GABA transporter (VGAT) (Synaptic Systems) and Alexa 647-conjugated goat anti-guinea pig (Invitrogen); and rabbit anti-cannabinoid receptor type 1 (CB1R) (Cayman) and Alexa 488-conjugated goat anti-rabbit (Invitrogen). All antibodies were used at 1 : 1000 dilution. Slices were imaged using a laser-scanning microscope (LSM 780; Carl Zeiss Germany) with a ×40 magnification and 1.0 NA oil and pinhole set at 1.2 µm for channels. The z-stack images (30 µm) were acquired at 0.6 µm optical sections and single midplane images were used for analysis. Perisomatic puncta quantification was performed using Puncta analyzer v2.0 in ImageJ (National Institute of Health) as previously described35 .
5
Immunohistochemistry of Drosophila Nervous System
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Unless stated otherwise, 3–5-day-old virgin female or male flies were dissected under phosphate-buffered saline (PBS; pH 7.4). Tissues were fixed for 30 min at room temperature in 4% paraformaldehyde in PBS. After extensive washing, the tissues were incubated in primary antibody for 48 h at 4 °C and in secondary antibody for 24 h at 4 °C. Antibodies used were: rabbit anti-GFP (1:1000; Invitrogen, A11122), mouse anti-GFP (1:1000; Sigma, G6539), rabbit anti-DsRed (1:1000; Clontech, 632496), mouse anti-nc82 (1:50; Developmental Studies Hybridoma Bank), Alexa 488-conjugated goat anti-mouse (1:1000; Invitrogen, A11001), Alexa 488-conjugated goat anti-rabbit (1:1000; Invitrogen, A11008), Alexa 568-conjugated goat anti-mouse (1:1000; Invitrogen, A11004), and Alexa 568-conjugated goat anti-rabbit (1:1000; Invitrogen, A11011). The CNS was mounted in Vectashield (Vector Laboratory, H-1000). Images were acquired with Zeiss LSM 700/Axiovert 200 M (Zeiss), and were processed in Image J55 (link).
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