Paraformaldehyde (PFA) fixed brains (4% in 0.1 M PBS) were washed in 0.1 M PBS, dehydrated in ethanol solutions with increasing concentrations (70, 80, 90, 96, 100%, three times, 30 min each), cleared in xylol (three times, 15 min and overnight), incubated (twice at 60 °C, overnight and 2 h), and finally embedded in paraffin. Sagittal brain slices (8 μm) were mounted on poly-l -lysine-coated glass slides. Paraffin was removed by incubation of the slices with RotI-histol (2 × 10 min, Carl Roth GMBH) and subsequent treatment with ethanol 100, 96, and 70% (3 min, each) and rinsing in H2O. Staining was performed in 0.1% cresyl violet solution for 5–10 min followed by short rinsing in H2O and two brief washes (in 96% ethanol). After dehydration in 100% ethanol (twice, 3 min each) slices were cleared in RotI-histol (twice, 3 min each) and mounted in a permanent mounting medium (Eukit, Sigma-Aldrich).
Roti histol
Manufactured by Merck Group
Sourced in Germany
RotI-histol is a piece of lab equipment designed for the purpose of rotating samples during histological processing. It is used to maintain consistent movement and mixing of samples during various stages of tissue preparation and staining.
Automatically generated - may contain errors
Lab products found in correlation
Roti histol, by Carl Roth (3 mentions)
Eukit, by Merck Group (1 mentions)
Hematoxylin gill no 3, by Merck Group (1 mentions)
Cellsens dimensions 2, by Olympus (1 mentions)
Eosin y, by Merck Group (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Dmi8 microscope, by Leica camera (1 mentions)
Microtome, by Leica camera (1 mentions)
Roti histofix, by Carl Roth (1 mentions)
Entellan mounting medium, by Merck Group (1 mentions)
4 protocols using roti histol
1
Cresyl Violet Staining of Brain Sections
2
Hematoxylin and Eosin Staining Protocol
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Specimens were stained for 3 min in hematoxylin (Gill No. 3, Sigma-Aldrich, Munich, Germany), washed 2 times with H2O followed by differentiating in acidic alcohol (1% HCl in ethanol) for 3 s and bluing under running tap water for 10 min. Counterstaining was conducted with eosin (Eosin Y, Sigma-Aldrich, Munich, Germany) for 10 s. Dehydration was performed in increasing alcohol series and Roti-Histol®. Finally, sections were mounted with Entellan®. One area per animal was evaluated based on the region of trauma. Six animals were used for each group based on the power analysis conducted for the animal experiment application (license number: 1183). Pictures were taken with the UC30 color camera at X10 and X40 magnification. All stainings were acquired with an Olympus IX81 and analyzed with the Olympus software cellSens Dimensions 2.3 (Build 18987).
3
Histological Analysis of Mouse Epididymal Adipose Tissue
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Histological analysis was performed on 5 μm paraffin sections of mouse epididymal adipose tissue. Paraffin sections were de-paraffinated with Roti®-Histol (Carl Roth) and rehydrated in ethanol baths with decreasing concentrations (100–30%). After washing with ultrapure water, Hematoxylin (Sigma-Aldrich) was applied for 3 sec. Tissue was dehydrated by ethanol baths (75% and 85%) before Eosin (Sigma-Aldrich) was applied for 3 sec. Tissue was further dehydrated by short baths in ethanol (95% and 100%), then treated with Roti®-Histol and fixed with Entallan®. Stained sections were imaged using a Leica DMi8 microscope with a HC PL Fluotar L 20x/0.40 objective and processed using the Leica LasX software and Fijii.133 (link) For determination of adipocyte diameter, Fijii with the PlugIn Adiposoft134 (link) was used. For determination of adipocyte area, the deep learning-based method Adipocyte U-Net132 (link) was used as published, except for setting the threshold for segmentation to 0.5 and excluding cells cut off by the picture frame.
4
Histological Analysis of Colon Tissue
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After the animals were sacrificed, adjacent colon tissue to the one used for MPM was preserved in Roti-Histofix (Carl Roth) and embedded in paraffin. Afterward, tissue samples were cooled at -20°C, and 3-µm slices were cut with a microtome (Leica) and collected on glass slides. The slides were then placed in an incubator at 65°C for at least an hour for the paraffin to melt and allow the tissue binding to the slide. The samples were then deparaffinized in Rotihistol (Carl Roth) and rehydrated in 100%, 96%, and 70% ethanol. Further, they were shortly washed in water and the cell nuclei were stained in Harris hematoxylin solution (Carl Roth). Counterstaining of cytoplasm was done by immersing the samples in eosin staining solution (Merck). The cuts were then dehydrated in 70%, 96%, and 100% ethanol. To complete the tissue dehydration, the slides were immersed in Rotihistol and mounted with Entellan mounting medium (Sigma Aldrich). The H&E stainings were then evaluated with the DMi4000B inverse microscope (Leica) at 10× and 20× magnification. Thus, the field of view in 2-dimensional H&E images was larger compared with the 3D stacks of MPM (25×, see the following).
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