Tissues were processed for cryosectioning or paraffin-embedding using standard methods. Kidney slices were stained with hematoxylin/eosin and PAS. Images were acquired using a bright field Aperto CS scanner and stored for subsequent analysis. Image Scope® software was used to measure glomerular size, cellularity and mesangial matrix expansion and deposition. A single-blind operator performed all analyses. Glomerular diameters were measured in all visible glomeruli in the histological section by taking the maximum diameter from the hilum to the end of the Bowman’s capsule. Glomerular cellularity and mesangial matrix deposition were assessed through a specific program quantified by Image Scope algorithm (Positive pixel count version 8.100). Frozen slices were pre-blocked with serum and incubated with the following antibodies: rat anti-CD31 (eBioscience) followed by donkey anti–rat FITC–conjugated antibodies (Jackson Immunology), rabbit anti-NG2 followed by donkey anti–rabbit Cy5–conjugated antibodies. Images were analyzed with a Leica confocal microscope (TCS-SP2) and by Image-J software (NIH, Bethesda, USA). Capillary density was quantified from at least five high-power fields (×40) per slide, two slides per animal (3 animals per group). Sections were randomly selected and analyzed under blinded conditions.
Rat anti cd31
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Rat anti-CD31 is a laboratory reagent used for the detection and identification of the CD31 (PECAM-1) protein in rat samples. CD31 is a cell surface glycoprotein expressed on endothelial cells, platelets, and certain leukocyte subsets. This antibody can be used in various applications, such as flow cytometry, immunohistochemistry, and Western blotting, to study the expression and distribution of CD31 in rat biological systems.
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Lab products found in correlation
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Hamster anti podoplanin, by Abcam (1 mentions)
Goat anti lyve 1, by R&D Systems (1 mentions)
Cm1850, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Imaris 8, by Oxford Instruments (1 mentions)
Spe confocal microscope, by Leica (1 mentions)
Rat anti endomucin, by Santa Cruz Biotechnology (1 mentions)
Mouse anti α sma, by Merck Group (1 mentions)
Rat anti ly6g, by BD (1 mentions)
Rabbit anti laminin, by Merck Group (1 mentions)
Rat anti pdgfrβ, by Thermo Fisher Scientific (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
Rat anti f4 80, by Abcam (1 mentions)
Cy3 or cy5 conjugated secondary antibodies, by Jackson ImmunoResearch (1 mentions)
Ds ri2, by Nikon (1 mentions)
Rabbit anti nf kb p65, by Cell Signaling Technology (1 mentions)
5 protocols using rat anti cd31
1
Quantitative Histological Analysis of Kidney
2
Immunofluorescent Analysis of Meningeal Vasculature
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Meninges were fixed overnight in 4% paraformaldehyde while still attached to the
skullcap. Skullcaps were then incubated in ethanol/acetone for 20 min at −20℃ and then the
meninges were carefully dissected away from the skullcap. Meninges were washed with PBS
and then blocked overnight with PBS + 0.3% TX-100 + 20% Aquablock (East Coast Bio, North
Berwick, ME, USA). Primary antibody was then added and the meninges were incubated
overnight at 4℃. The following primary antibodies were used: rat anti-CD31 (1:1000;
eBiosciences, San Diego, CA, USA), goat anti-Lyve-1 (1:1000; R&D Systems, Minneapolis,
MN, USA), hamster anti-podoplanin (1:1000; Abcam, San Francisco, CA, USA), and rabbit
anti-Prox1 (1:500; AngioBioCo, Del Mar, CA, USA). Tissues were washed with PBS + 0.3%
TX-100 and then incubated overnight at 4℃ with the appropriate secondary antibodies.
Tissues were washed again with PBS + 0.3% TX-100, placed on slides, and coverslips were
mounted with ProLong Gold (Thermo Fisher Scientific, Waltham, MA, USA) anti-fade
reagent.
skullcap. Skullcaps were then incubated in ethanol/acetone for 20 min at −20℃ and then the
meninges were carefully dissected away from the skullcap. Meninges were washed with PBS
and then blocked overnight with PBS + 0.3% TX-100 + 20% Aquablock (East Coast Bio, North
Berwick, ME, USA). Primary antibody was then added and the meninges were incubated
overnight at 4℃. The following primary antibodies were used: rat anti-CD31 (1:1000;
eBiosciences, San Diego, CA, USA), goat anti-Lyve-1 (1:1000; R&D Systems, Minneapolis,
MN, USA), hamster anti-podoplanin (1:1000; Abcam, San Francisco, CA, USA), and rabbit
anti-Prox1 (1:500; AngioBioCo, Del Mar, CA, USA). Tissues were washed with PBS + 0.3%
TX-100 and then incubated overnight at 4℃ with the appropriate secondary antibodies.
Tissues were washed again with PBS + 0.3% TX-100, placed on slides, and coverslips were
mounted with ProLong Gold (Thermo Fisher Scientific, Waltham, MA, USA) anti-fade
reagent.
3
Neutrophil Distribution in Parabiotic Mice
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We analyzed partner-derived neutrophils distribution in tissues of WT mice that were maintained in parabiosis with Ly6GTOM for 8 wk. Tissues were collected and frozen in OCT, and 30-µm slices were obtained in a cryostat (CM1850; Leica). Tissue sections were incubated with rat anti-endomucin (Santa Cruz) and rat anti-CD31 (eBioscience), followed by incubation with DAPI (Life Technologies), the endothelial-binding lectin IB4 conjugated with AF647 (Thermo Fisher), and a secondary chicken anti-rat AF647 antibody (Molecular Probes). Images from Ly6GTOM+ neutrophils were obtained using an SPE confocal microscope (Leica). 3D images of neutrophils and vessels were reconstructed with Imaris 8.0 (Bitplane AG). Quantification of intra- and extravascular neutrophils was done manually.
4
Multiparametric Kidney Histology Analysis
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Mice were euthanized under deep isoflurane anesthesia and immediately perfused via the left ventricle with ice-cold PBS for 1 min. Kidneys were harvested, cut in transversal sections, fixed in freshly prepared 4% paraformaldehyde and 5 μm cryosections (Tissue Tek, Sakura) were stained using the following primary antibodies: rat anti-F4/80 (Abcam, 1:1000), rat anti-CD31 (eBioscience, 1:600), rabbit anti-CD3 (eBioscience, 1:300), rabbit anti-laminin (Sigma, 1:2000), rat anti-PDGFRβ (eBioscience, 1:800), mouse anti-αSMA (Sigma, 1:2500), rat anti-Ly6g (BD Pharmingen, 1:500). Sections were subsequently incubated with corresponding Cy3- or Cy5-conjugated secondary antibodies (Jackson ImmunoResearch, 1:400). For PAS staining, kidney sections were fixed in formalin. Images were captured using Nikon C1 confocal and Zeiss Axio Observer Z1 microscope, respectively.
5
Immunofluorescence Imaging of Skin Cells
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Fresh skin tissues from mice were embedded with OCT (Tissue Tek). Skin sections (6 μm) and HaCaT cells grown on round glass coverslips in 24-well plates were fixed with 4 % paraformaldehyde (PBS) for 10 min. Sections were blocked with 5 % donkey serum for 1 h after permeabilization with 0.3 % Triton X-100 for 15 min at room temperature. Primary antibodies were incubated overnight at 4 °C. After washing with PBS, Alexa Fluor 488-conjugated or 594-conjugated secondary antibody (Thermo Fisher Scientific) was added for 1 h at room temperature. Sections were washed and counterstained with 4′, 6-diamidino-2-phenylindole (DAPI). All images were captured with a Nikon DS-Ri2 (Nikon, Japan). The following primary antibodies were used: Rat anti-CD4 (1:100, eBioscience), Rat anti-CD31 (1:100, eBioscience), Rabbit anti-Ki67 (SolA15) (1:500, eBioscience™, #PA5-114437), Rabbit anti-NF-kB p65 (1:100, Cell Signaling) antibodies.
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