Computational protocol: Vascular Endothelial Growth Factor (VEGF) Bioavailability Regulates Angiogenesis and Intestinal Stem and Progenitor Cell Proliferation during Postnatal Small Intestinal Development

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[…] Formalin-fixed tissue was embedded in paraffin and sectioned at 5 μm. The tissue was dehydrated in 30% EtOH for 30 minutes, 50% EtOH for 45 minutes, 70% EtOH for 45 minutes, 95% EtOH for 1 hour, 100% EtOH for 1 hour twice. The tissue was then cleared with toluene twice for 45 minutes, then in 1:1 ratio of paraffin and toluene overnight at 65°C. The following morning the tissue was placed in pure paraffin for 1 hour twice and fixed in the cassette on a cooling plate to solidify. The tissue was then sectioned at 5 μm. Frozen sections were procured for confocal microscopy to evaluate angiogenesis after intracardiac FITC-dextran labeling of the vasculature. Tissue was protected from light and placed in 4% paraformaldehyde overnight at 4°C. The following day, tissue was placed in 30% sucrose until it sank to the bottom the tube. The tissue was then transferred to a 1:1 mixture of 30% sucrose:OTC compound overnight at 4°C. Tissue was subsequently transferred to OTC compound and slowly frozen at -20°C until the OTC was frozen. Blocks were stored at -80°C until sectioning. Frozen tissue was sectioned at 60 μm immunofluorescence staining with DAPI was performed as describe below.Hematoxylin and eosin (H&E) staining was performed on the sectioned tissue. Slides were placed in Histochoice (Amresco; H103) twice for 2 minutes, then in 100% EtOH for 2 minutes. The slides were hydrated in 70% EtOH for 30 seconds, 50% EtOH for 30 seconds, 30% EtOH for 30 seconds, and H2O for 2 minutes. The slides were placed in hematoxylin for 15 seconds and H2O until clear, followed by eosin for 5 seconds for counterstain. The slides were dehydrated in 90% EtOH for 1 minute, 100% EtOH for 1 minute and Histochoice for 2 minutes. The slides were imaged at 20x magnification on a brightfield microscope. Single and multiple clusters of red blood cells (RBCs) were counted by blind observers as previously described []. The villus length, crypt depth, circumference and number of crypts per intestinal length were measured by a trained blinded observer with ImageJ [].Immunofluorescence was performed to locate terminal cell markers and proliferative markers. The slides were placed in Histochoice for 10 minutes twice. The slides were rehydrated by soaking them sequentially in 100% EtOH, 90% EtOH, 75% EtOH, 50% ETOH, and 30% EtOH for 5 minutes each. A low pH, citrate-based Antigen Unmasking Solution (Vector; H-3300) was used to retrieve antigens in the microwave. The microwave heated the solution for 4 minutes at 50% three times, with 30 seconds in between each heating session. The solution was cooled to room temperature for 30 minutes and washed in phosphate-buffered saline (Gibco; Cat#10010) with 0.1% Tween (Amresco; Cat# 9005-64-5) (PBS-T) for 5 minutes. Universal blocking solution (1% BSA, 0.1% cold fish skin gelatin, 0.5% Triton-X 100 and 1x PBS) with 2% goat serum (Sigma-Aldrich; Cat# G9023) was applied for 30 minutes at room temperature. Primary antibodies were diluted () in universal blocking solution with 2% goat serum and placed on tissue overnight at 4°C. In the morning, tissues were washed in PBS-T for 5 minutes three times. Appropriate Cy3 or Cy5 secondary antibodies diluted in PBS with 0.05% Tween () and applied to tissue for 1 hour at room temperature. The slides were again washed with PBS-T for 5 minutes three times. The sections were mounted on Vectashield with DAPI (Vector; Cat# H-1200) and visualized under the fluorescent microscope.Lysozyme was quantified by percentage of immunofluorescent-positive cells per hemi-crypt by a trained, blinded observer with ImageJ. Ki-67-positive cells were counted in the crypt and amplifying zone by the number of positive cells per position by a trained, blinded observer with ImageJ. [...] VEGF mutant and littermate duodenal cross sections were prepared as described above and imaged for FITC and DAPI immunofluorescence using a Zeiss LSM 710 confocal microscope. Sixty micron sections were imaged at an individual z stack thickness of 1 μm to allow for accurate three-dimensional volume reconstruction using IMARIS software. Confocal images were imported into IMARIS version 7.7.2 and volume reconstruction analysis was performed using region of interest selection of FITC-labeled vasculature of individual hemivilli. Threshold values were obtained from littermate controls using autothresholding algorithms and these values were applied to VEGF mutants to allow for proper comparison of three-dimensional volume analysis. Sixty villi in total from four individual mice per group were analyzed. […]

Pipeline specifications

Software tools ImageJ, Imaris
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Drosophila melanogaster
Diseases Intestinal Diseases
Chemicals Doxycycline