Mice were sacrificed 24 hours following fibrin clot placement, and the spleens and lungs were harvested for histological evaluation. The tissues were fixed in either 10% formalin or methyl Carnoy’s solution, processed, embedded in paraffin, and sectioned (5 μm). Tissues were immunostained with F4/80 (AbD Serotec) or Ly6G (BioLegend) antibodies, for assessment of macrophage or neutrophil infiltration respectively, and scored by an investigator who was blinded to the group. The area of positively stained cells was calculated per 40X objective using FRIDA Software (FRamework for Image Dataset Analysis, [40 (link)]), and numerous random fields were assessed per tissue section. Immunostaining using an isotype control antibody was also performed to demonstrate a lack of non-specific staining (Supplemental Figure 1 ).
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Histological Analysis of Immune Cell Infiltration
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Antibodies
Antibody
Clot
Embedded paraffin
Fibrin
Formalin
Isotype
Lungs
Macrophage
Mice
Neutrophil infiltration
Tissue
Corresponding Organization : Harvard University
Other organizations : Albert Einstein College of Medicine, Washington State University, National Health Research Institutes, Boston University
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Variable analysis
independent variables
- Fibrin clot placement
- Macrophage infiltration
- Neutrophil infiltration
- Time (24 hours) following fibrin clot placement
- Tissue fixation method (10% formalin or methyl Carnoy's solution)
- Tissue processing (embedded in paraffin, sectioned at 5 μm)
- Immunostaining (F4/80 antibody for macrophages, Ly6G antibody for neutrophils)
- Evaluation by a blinded investigator
- Immunostaining using isotype control antibody to demonstrate lack of non-specific staining
- Not explicitly mentioned
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