1. Histological Analysis<ol><li>Remove small intestine samples from each experimental rat by surgical procedure. Fix samples in Bouin&#8217;s Fixative, embed in paraffin, prepare 6 &#181;m sections and stain sections with hematoxylin and eosin using standard procedures.</li><li>Use a high-resolution microscope system for measuring height of intestinal villi and total mucosal thickness in each sample (magnification 100X). Analyze 4 samples from each rat and make at least twenty measurements in each sample. Express results in micrometers.</li></ol>2. High-Resolution Light Microscopy of Blood<ol><li>Use a vibration isolation platform as a base for the microscope system and video camera and computer to record the live images. Calibrate test images using a Richardson slide.</li><li>Place 7 &#181;l of freshly drawn blood from each rat on a glass slide, coverslip, photograph and record ten image frames of 72 &#215; 53.3 &#181;m2 in each sample (magnification 1,700X).</li><li>Measure the concentration of vesicles using software that provides high-resolution direct-view optical images in real time. Examine a minimum of 20 image frames for each experimental condition.</li></ol>

<table><tbody><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich, St. Louis, MO</td><td>P4417</td><td> </td></tr><tr><td>Bouin&#8217;s Fixative</td><td>Electron Microscopy Sciences, Hatfield, PA, USA</td><td>15990</td><td> </td></tr><tr><td>Richardson test slide</td><td>Electron Microscopy Science, Hatfield, PA, USA</td><td>80303</td><td> </td></tr><tr><td>Sony DXC-33 Video camera</td><td>Sony</td><td> </td><td> </td></tr><tr><td>Light microscope optical system</td><td>CitoViva Technology Inc., Auburn,AL</td><td> </td><td> </td></tr><tr><td>Image-Pro Plus software</td><td>Media Cybernetics, MD, USA</td><td> </td><td> </td></tr></tbody></table>

histological and hematological analyses to evaluate the effects of bacillus subtilis in heat-stressed rats

Source: <a href='https://app.jove.com/v/54122/prevention-heat-stress-adverse-effects-rats-bacillus-subtilis'>Sorokulova, I., et al. Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain. J. Vis. Exp. (2016)</a>This video presents histological and blood microscopy analyses used to evaluate the protective effects of Bacillus subtilis in rats exposed to heat stress.

Histological and Hematological Analyses to Evaluate the Effects of Bacillus subtilis in Heat-Stressed Rats

Source: Sorokulova, I., et al. Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain. J. Vis. Exp. (2016)This video presents ...

Begin with hematoxylin and eosin-stained intestinal sections from heat-stressed rats, one orally pre-treated with buffer as a control, and the other with a Bacillus subtilis suspension.Heat stress damages intestinal tight junction proteins, resulting in shortened villi and mucosal thinning.Pre-treatment with the bacteria promotes the release of signaling molecules that stabilize tight junction proteins, preserving villus height and mucosal thickness.Observe the sections under the high-resolution light microscope to identify differences in villus height.Next, place a drop of fresh blood from each rat on glass slides and cover with coverslips.Heat stress destabilizes erythrocyte membranes, releasing microvesicles into the bloodstream.The bacteria reduce systemic oxidative stress, stabilizing erythrocyte membranes and minimizing vesiculation.Observe the blood samples under the high-resolution light microscope to assess differences in vesiculation.Preserved villus height in the intestinal section and fewer microvesicles in the blood confirm the protective effect of the bacteria under heat stress.After preparing and staining sections of the small intestines according to the text protocol, use a high-resolution microscope to measure the intestinal villi and total mucosal thickness. Analyze at least four samples from each rat and make at least 20 measurements in each sample.To carry out high-resolution light microscopy of blood samples, use the vibration isolation platform as a base for the microscope system with a video camera and computer to record live images. After calibrating test images according to the text protocol, place seven microliters of freshly drawn blood from each rat on a glass slide and add a coverslip. Then, photograph and record ten image frames of 72 by 53.3 micrometers squared in each sample.Finally, using software that provides high-resolution direct-view optical images in real time, measure the concentration of vesicles.

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274 Views. Begin with hematoxylin and eosin-stained intestinal sections from heat-stressed rats, one orally pre-treated with buffer as a control, and the other with a Bacillus subtilis suspension.Heat stress damages intestinal tight junction proteins, resulting in shortened villi and mucosal thinning.Pre-treatment with the bacteria promotes the release of signaling molecules that stabilize tight junction proteins, preserving villus height and mucosal thickness.Observe the sec...

Histological and Hematological Analyses to Evaluate the Effects of Bacillus subtilis in Heat-Stressed Rats

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Name	Company	Catalog Number
Phosphate buffered saline (PBS)	Sigma-Aldrich, St. Louis, MO	P4417
Bouin’s Fixative	Electron Microscopy Sciences, Hatfield, PA, USA	15990
Richardson test slide	Electron Microscopy Science, Hatfield, PA, USA	80303
Sony DXC-33 Video camera	Sony
Light microscope optical system	CitoViva Technology Inc., Auburn,AL
Image-Pro Plus software	Media Cybernetics, MD, USA