Blood smears on poly-L-lysine-coated glass slides were fixed in 100% methanol, air-dried, fixed with 4% paraformaldehyde, and blocked and permeabilized (2% goat serum + 0.1% Triton X-100) for 4 hours at room temperature. Slides were then incubated with anti-neutrophil elastase rabbit pAb (25 μg/mL; Calbiochem) and anti-H2A-H2B-DNA mouse mAb (clone PL2-6; 1 μg/mL ; gift of Marc Monestier, Temple University, Philadelphia, PA) in 0.3% bovine serum albumin overnight at 4 °C. Next, the slides were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 555-conjugated goat anti-mouse IgG (both 1:500; Life Technologies) for 45 minutes at room temperature, rinsed with PBS, and mounted using VECTASHIELD Mounting Medium with DAPI (Vector Laboratories). Images were captured with a QImaging Retiga 2000R camera using a Nikon S Plan Fluor ELWD 60 × /0.70 objective on a Nikon Eclipse 90i microscope.
Retiga 2000r
Manufactured by Nikon
The Retiga 2000R is a scientific-grade digital camera designed for microscopy and imaging applications. It features a high-resolution CCD sensor and a range of customizable settings to capture detailed, high-quality images. The core function of the Retiga 2000R is to provide reliable and accurate image acquisition for various scientific and laboratory-based research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
S plan fluor elwd, by Nikon (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Eclipse 90i microscope, by Nikon (1 mentions)
Smz1500, by Nikon (1 mentions)
Nis elements ar 4, by Nikon (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Dab kit, by Vector Laboratories (1 mentions)
Cytoseal 60 mounting medium, by Thermo Fisher Scientific (1 mentions)
Eclipse ni u microscope, by Nikon (1 mentions)
Eclipse e600 microscope, by Nikon (1 mentions)
4 protocols using retiga 2000r
1
Immunofluorescent Staining of Neutrophils
2
Birefringence Assay for Zebrafish Myofiber Analysis
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The sapje/sapje-like dystrophic muscle phenotype was detected by using a birefringence assay, a technique used to analyze myofiber integrity using polarized light performed as described previously (Granato et al., 1996 (link)). Polarizing filters were placed on a bottom-lit dissection scope, and images were acquired with a QImaging Retiga 2000R camera fitted to a Nikon SMZ1500 microscope using OpenLab software. Zebrafish were anesthetized with tricaine and positioned relative to the polarized light to produce maximal birefringence illumination.
3
Immunohistochemistry Analysis of Pathology Spreading
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We stained coronal free-floating sections using primary antibodies and secondary biotinylated antibodies listed in Table S5 . For the detection of the antibody with DAB, we used a standard peroxidase-based method (Vectastain ABC kit and DAB kit; Vector Laboratories). Sections stained for human α-syn were then counterstained by hematoxylin. After dehydration, slides are coverslipped with Cytoseal 60 mounting medium (Thermo Fisher Scientific). We analyzed sections with conventional light using an Eclipse Ni-U microscope (Nikon); images were captured with a Retiga 2000R digital camera using NIS Elements AR 4.00.08 software (Nikon).
For analysis of pathology spreading in the brains of mice, we stained with anti-pser129 antibody a whole series of coronal sections (210-µm intervals between consecutive sections) from four to five animals per group (noninjected, PBS, mMs, mPFFs, and HuPFFs injected groups). We assessed in a blinded manner the presence of pser129-positive accumulations by screening every single section at 20× magnification using an Eclipse Ni-U microscope (Nikon).
For analysis of pathology spreading in the brains of mice, we stained with anti-pser129 antibody a whole series of coronal sections (210-µm intervals between consecutive sections) from four to five animals per group (noninjected, PBS, mMs, mPFFs, and HuPFFs injected groups). We assessed in a blinded manner the presence of pser129-positive accumulations by screening every single section at 20× magnification using an Eclipse Ni-U microscope (Nikon).
4
Immunohistochemical Analysis of α-Synuclein
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