Computational protocol: Three-dimensional structure and cytokine distribution of platelet-rich fibrin

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Protocol publication

[…] The PRF sample was fixed, dehydrated and placed in a tissue cassette and embedded in paraffin wax. Infiltration of paraffin into the porous tissue maintains the intrinsic morphology of the tissue and solidifies the sample to allow fine sectioning. The sample was divided into seven segments (), then cut into 3–5 μm sections for histological analyses. The RBC end and plasma end of the PRF were similar and easily confused during this preparation of histological samples. Sample orientation errors were avoided by labelling. The tissue sections were mounted on slides, stained with haematoxylin-eosin or Masson’s trichrome stain, viewed and photographed under an optical microscope . The compactness of the fibrin network (i.e., porosity, [n]) was measured directly by scanning electron microscopy (SEM) using ImageJ software (NIH, Bethesda, MD, USA). Porosity [n]=Ap/At × %, where Ap denotes the measured total area of the pores in each SEM image and At is the total area of an SEM image.All protocols of histological and SEM analyses for the animal rabbit model were developed for this PRF sample and were slightly modified from the typical tissue preparation method for light microscopy. Optical microscopy and SEM were used in a complementary fashion, as they have different levels of resolution for inspecting the fibrin microstructure (the resolution of the optical microscope falls within the μm range and that of the SEM can be in nm). To prevent charge accumulation and damage to tissue sections during SEM examination, approximately 80 Å of platinum was deposited on the tissue sections using a sputter coater. SEM images were acquired using a field-emission scanning electron microscope (FESEM, Model JSM-5600, JEOL, Tokyo, Japan) operated at an accelerating voltage of 15 kV. Serial images were aligned sequentially in three-dimensional space and visualized three-dimensionally with Fiji and TrakEM2 software ,. In a typical procedure, serial slides can be manually aligned by setting up a number of pairs of corresponding control points to the same (x, y) location for consecutive images zi and zi+1, and the pairs of images and paired-sets of control points are then given to semi-automatic software for image alignment. Fully automatic registration of biological images is then possible as demonstrated by the software - TrakEM2 ,. The average diameters of the fibrin fibres were obtained by averaging diameters directly measured in the SEM images of ∼30 samples. […]

Pipeline specifications

Software tools ImageJ, TrakEM2
Applications cryo-EM, Microscopic phenotype analysis
Organisms Oryctolagus cuniculus, Homo sapiens