MEFs were pulse-labelled with 25 uM CldU for 20 min, washed three times with medium, incubated in 2 mM HU for 4 hours, washed three times with medium, and pulse-labelled with 250 uM IdU for 20 min. Labelled cells were harvested, lysed in spreading buffer (200 mM Tris-HCL ph 7.4, 50 mM EDTA, 0.5% SDS) and DNA fibers were spread on slides. CldU and IdU were detected by incubating acid-treated fiber spreads with rat anti-BrdU monoclonal antibody (AbD Serotec) and mouse anti-BrdU monoclonal antibody (Becton Dickinson) for 1 hr at 37°C respectively. Slides were fixed with 4% PFA and incubated with AlexaFluor 555–conjugated goat anti–rat IgG (Molecular Probes) and AlexaFluor 488–conjugated goat anti–mouse IgG (Molecular Probes) for 1 hr at 37°C. Fibers were imaged using an LSM780 (Carl ZeissMicroscopy Ltd) confocal microscope and analysed using ImageJ software (NIH). At least 100 tracks were scored per condition.
Alexafluor 555 conjugated goat anti rat igg
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan
AlexaFluor 555–conjugated goat anti–rat IgG is a secondary antibody reagent that binds to rat immunoglobulin G (IgG) and is labeled with the AlexaFluor 555 fluorescent dye. It is commonly used in immunofluorescence assays, flow cytometry, and other applications that require the detection of rat-derived antibodies or antigens.
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Lab products found in correlation
Alexa fluor 488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (3 mentions)
Mouse anti brdu monoclonal antibody, by BD (3 mentions)
Rat anti brdu monoclonal antibody, by Bio-Rad (3 mentions)
Radiance confocal microscope, by Bio-Rad (2 mentions)
Lsm 700, by Zeiss (1 mentions)
Goat serum, by Nichirei Biosciences (1 mentions)
Bacterial protease subtilisin a, by Merck Group (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
6 protocols using alexafluor 555 conjugated goat anti rat igg
1
DNA Fiber Assay for Replication Dynamics
2
DNA Replication Tracking with CldU and IdU
3
DNA Fiber Analysis of Replication Dynamics
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MCF7 cells were transfected with 100 nM hSSB1 siRNA or control siRNA (Allstars negative control siRNA, Qiagen) using Lipofectamine 2000 according to manufacturer's instructions, and re-transfected with hSSB1 siRNA 24 h later. Twenty-four hours after the second transfection, cells were pulse labelled with 25 μM CldU for 20 min, washed three times with medium, incubated in 2 mM HU for 2 h, washed three times with medium and pulse labelled with 250 μM IdU for 1 h. Labelled cells were harvested and DNA fibre spreads prepared as previously described (23 (link)). CldU was detected by incubating acid treated fibre spreads with rat anti-BrdU monoclonal antibody (1:1000, AbD Serotec) for 1 h. Slides were fixed with 4% PFA (paraformaldehyde) and incubated with Alexa-Fluor 555-conjugated goat anti-rat IgG (1:500, Molecular Probes) for 1.5 h. IdU was detected using mouse anti-BrdU monoclonal antibody (1:1000, Becton Dickinson) over night at 4°C and Alexa-Fluor 488-conjugated goat anti-mouse IgG (1:500, Molecular Probes) for 1.5 h. Fibres were examined using a Biorad Radiance confocal microscope with a 60× oil immersion objective. For quantification of replication structures, at least 250 structures per experiment were counted using the ImageJ software (National Institutes of Health, http://rsbweb.nih.gov/ij/ ). Statistical analysis was performed using a one-tailed paired t-test.
4
Immunofluorescence Staining of Giardia
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Giardia cells were applied to the poly-L-lysine coated glass slides for 10 min, fixed in ice-cold methanol at –20°C for 10 min, and permeabilized with phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4) with 0.5% Triton X-100 for 10 min. After blocking for 1 h in PBS/5% goat serum/3% bovine serum albumin, the cells were incubated with 1:100 diluted anti-GlKin-13 antibodies or anti-phosphorylated PLK (ab39068; Abcam, Cambridge, Massachusetts, USA) overnight at 4°C, and subsequently incubated with 1:200 diluted Alexa Fluor 555-conjugated goat anti-rat IgG (Thermo Fisher Scientific). The slides were counterstained with diamidino-2-phenylindole (DAPI) and observed using an inverted confocal laser scanning microscope (LSM700; Carl Zeiss, Oberkochen, Germany).
5
Immunofluorescence Detection of Gd-IgA1 in Renal Biopsies
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Immunofluorescence staining of Gd-IgA1 in renal biopsy specimens was conducted using formalin-fixed, paraffin-embedded sections of 3 μm in thickness. After deparaffinization using xylene and ethanol concentration series and subsequent rehydration, antigen retrieval using 0.05% bacterial protease subtilisin A (Sigma-Aldrich, Tokyo, Japan) dissolved in phosphate-buffered saline (PBS) was performed at room temperature for 2 h. Samples were then rinsed with distilled water 3 times and blocked with 10% goat serum (Nichirei Bioscience, Tokyo, Japan) at room temperature for 30 minutes, followed by incubation with KM55Ⓡ antibody (Immuno-Biological Laboratories, Fujioka, Japan) at 37°C for 60 minutes. After 3 washes with PBS, samples were incubated with Alexa Fluor 555-conjugated goat anti-rat IgG (1:1,000; Thermo Fisher, Tokyo, Japan) at 37°C for 30 minutes. Samples were washed with PBS and incubated with FITC-conjugated polyclonal goat anti-human IgA (1:40; MBL, Nagoya, Japan) at 37°C for 30 minutes. After being washed again with PBS, the slides were sealed. Tissues were observed under an LSM710 immunofluorescent microscope (Carl Zeiss, Oberkochen, Germany). The specificity of KM55Ⓡ antibody against Gd-IgA1 was previously demonstrated by immunohistochemistry and a neutralization assay (9 (link)).
6
Immunofluorescence Localization of Giardia Proteins
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Giardia cells were attached to glass slides coated with l -lysine for 10 min, followed by treating with ice-cold methanol for 10 min and permeabilizing with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10.1 mM Na2HPO4 and 2 mM KH2PO4, pH 7.4)/0.5% Triton X-100 for 10 min. After blocking for 1 h in PBS/5% goat serum/3% bovine serum albumin (BSA), the cells were incubated with the primary antibodies in PBS/3% BSA overnight at 4 °C and treated with fluorescent dye-conjugated secondary antibodies afterwards. The samples were mounted with ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific) and observed using an inverted confocal laser scanning microscope (LSM700; Carl Zeiss, Oberkochen, Germany). The following antibodies were used at the indicated dilutions: anti-SpCas9 mouse monoclonal antibodies (1:100; Abcam), rat anti-GlCWP1 polyclonal antibodies (1:200; [32 (link)]), Alexa Fluor 488-conjugated goat anti-mouse IgG (1:100; Thermo Fisher Scientific), and Alexa Fluor 555-conjugated goat anti-rat IgG (1:200; Thermo Fisher Scientific).
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