To determine the localization of Cs1 in C. sinensis adult worms, they were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced at 6–7 μm thicknesses. Paraffin sections were deparaffinized, rehydrated, and then restoration heat-treated. The sections were incubated with anti-rCs1 mAb (Cs1-2-6-3) diluted 1:20 in PBS for 2 h at 37°C, with SP2/0 used as a negative control. The sections were washed three times with PBS and then incubated with fluorescein isothiocyanate-conjugated anti-mouse IgG (Sigma, USA) for 1 h at 37°C in the dark. After washing, sections were observed under a fluorescence microscope (Olympus BX51).
Fluorescein isothiocyanate conjugated anti mouse igg
Manufactured by Merck Group
Sourced in United States
Fluorescein isothiocyanate-conjugated anti-mouse IgG is a laboratory reagent used for the detection and analysis of mouse immunoglobulin G (IgG) in various experimental techniques. It consists of fluorescein isothiocyanate (FITC), a fluorescent dye, coupled to antibodies specific to mouse IgG. This product provides a means to visualize and identify the presence of mouse IgG in samples.
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3 protocols using fluorescein isothiocyanate conjugated anti mouse igg
1
Localization of Cs1 in C. sinensis
2
Viral Isolation and Characterization
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Fluorescent antibody testing and negative staining EM were performed to confirm viral isolation. Fluorescent antibody signals and visible viral particles on EM images provided positive evidence of ARV isolation. BSR cells were used to perform fluorescent antibody tests. The monoclonal antibody 1F4 against σC, which was produced in our laboratory, was used as the primary antibody and fluorescein isothiocyanate-conjugated anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA) was used as the secondary antibody. Stained particles were observed under a fluorescent inverted microscope (AMG EVOS™ f1, Thermo Fisher Scientific).
Viral morphology was observed via EM to confirm the presence of viral particles. The preserved suspensions from the 11 isolates were centrifuged at 12000 × g at 4 °C for 30 min. Particles were resuspended in sterile PBS before EM analysis.
Viral morphology was observed via EM to confirm the presence of viral particles. The preserved suspensions from the 11 isolates were centrifuged at 12000 × g at 4 °C for 30 min. Particles were resuspended in sterile PBS before EM analysis.
3
Sperm DNA Fragmentation Assay
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Immunodetection of acetylated tubulin allowed the visualization of flagella. Semen samples smeared onto glass slides were incubated with horse serum (1:80 in PBS, Sigma) for 30 min at room temperature. After a 5-min wash in 4 × standard saline citrate (SSC) containing 0.05% Tween-20 (SSC-Tween), anti-acetylated tubulin mouse antibody (1:100 in PBS, clone 6-11B-1, Sigma) was added for 1 h at room temperature. Samples were then washed for 5 min in SSC-Tween and incubated with fluorescein isothiocyanate-conjugated antimouse IgG (1:100 in PBS, Sigma) for 15 min at 37°C. After two 5-min washes in SSC-Tween and a post-fixation in methanol for 2 h at -20°C, (TdT)-mediated dUTP nick-end labelling (TUNEL) assays were performed to analyse DNA fragmentation using the In situ Cell Death Detection kit POD ® (Roche, Mannheim, Germany) according to the manufacturer's instructions. Slides were counterstained with propidium iodide diluted 1:1000 in antifading mounting medium (Antifade ® , MP-QbioGene, Illkirch, France). DNA fragmentation was characterized on 500 spermatozoa at a ×1000 magnification. Spermatozoa were considered to have fragmented DNA when the green fluorescence signal occupied >50% of head area.
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