Following deparaffinization, slides were stained in Hematoxylin (Sigma Aldrich) for 3 min. The excess of Hematoxylin stain was removed by short immersion of slides in 1% HCl acid solution followed by another short immersion in 0.1% LiCO3 solution. Samples were then stained with Eosin Y solution (Sigma Aldrich) for 30 s and dehydrated using 70, 90, and 100% ethanol for 30 s each, followed by xylene for 10 min. Slides were mounted with Entellan mounting medium (ProSciTech) and dried for 1 h. Images were obtained using Olympus BX-51 upright bright-field microscope.
Bx51 upright brightfield microscope
Manufactured by Olympus
The BX51 is an upright brightfield microscope designed for general laboratory use. It features a stable and durable construction, and provides clear and consistent optical performance. The microscope is equipped with a range of objective lenses to enable viewing of samples at different magnification levels.
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5 protocols using bx51 upright brightfield microscope
1
Hematoxylin-Eosin Staining Protocol
2
Detailed Necropsy and Histological Analysis
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At necropsy, all organs, vasculature supply to organs, skeletal muscle, and major blood vessels were evaluated in all animals immediately after euthanasia. The resected liver length (RL), width, and mass (RM) were measured. The actual percent hepatectomy was calculated from the RL/LL × 100. The transected liver was examined for number of vessels transected, the diameter of the largest vessel transected, and cross-sectional area of identifiable vessels. Representative sections of liver, kidney, heart, lung, small intestine, and mesenteric lymph nodes were collected for histological evaluation. Tissues were fixed, stained with hematoxylin and eosin, and then examined by a pathologist using an Olympus BX51 upright brightfield microscope. The measured length of the resected portion of the left lobe was compared with the attempted length of resection to assess for consistency and accuracy of intended hepatectomy.
3
Histological Analysis of Brain Tissue
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Following deparaffinization, brain tissue was stained in Haematoxylin (Sigma Aldrich) for 3 min and the excess of the stain was removed by quick immersion of slides in 1% HCl solution, followed by another short immersion of slides in 0.1% LiCO3 solution. Next, slides were stained with Eosin Y solution (Sigma Aldrich) for 30 s and dehydrated in a series of ethanol dilutions (70, 90, and 100% ethanol for 30 s each) and xylene for 10 min. Slides were mounted with Entellan mounting medium (ProSciTech) and dried for 2 hours at room temperature. Images were obtained using Olympus BX-51 upright bright-field microscope. Whole-brain images were acquired as tiled image stacks. Cortical layers thickness relative to section thickness and cerebellar area relative to whole-brain area were measured using ImageJ software88 (link). Measurements of length and area are indicated by lines on the respective figures.
4
Histopathologic Evaluation of Tissues
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At time of euthanasia, necropsy was performed to collect representative sections of brain (hippocampus, cerebellum, frontal cortex), heart, lungs, liver, kidney, spleen, ileum, adrenal glands, and mesenteric lymph nodes for histopathologic evaluation. Tissue sections were fixed and stained with H&E, and then examined by a veterinarian pathologist blinded to the experimental groups, using an Olympus BX51 upright brightfield microscope (Olympus Scientific Solutions, Waltham, MA). Tissues were scored for severity of pathology as detailed below (table 4).
5
Histopathological Analysis of C. rodentium Infection
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At necropsy on day 14 post-infection, ceca and large intestines of either mock- or C. rodentium-infected animals were collected, placed in cassettes, and fixed in 10% buffered formalin for 24 h. They were then dehydrated and embedded in paraffin, and 5-μm tissue sections were cut and stained with hematoxylin and eosin (performed by The University of Texas Medical Branch Department of Pathology). Slides were visualized for pathology using an Olympus BX51 upright brightfield microscope.
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