Patient-derived OA chondrocyte pellets were fixed dehydrated and embedded in paraffin. Paraffin was removed using xylol, isopropanol, and an alcohol gradient in descending order (all from Carl Roth, Karlsruhe, Germany). Sections were stained for 2 min in Mayer’s Hematoxylin solution (Carl Roth) followed by a washing step in tap water. Subsequently, sections were incubated in 50% and 70% ethanol for 2 min respectively and counterstained in Eosin solution (Carl Roth) for 45 s. Afterwards sections were dehydrated using an ascending alcohol gradient followed by xylol incubation. Sections were mounted using entellan (Carl Roth).
Eosin solution
Manufactured by Carl Roth
Sourced in Germany
Eosin solution is a commonly used dye in histology and cytology. It is an anionic xanthene dye that binds to basic structures in cells, staining them pink or red. The solution is available in various concentrations and is typically used as a counterstain in combination with other dyes, such as hematoxylin, to provide contrast in microscopic examinations of tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Hematoxylin solution, by Carl Roth (2 mentions)
Mayer s hematoxylin solution, by Carl Roth (1 mentions)
Haematoxylin solution, by Thermo Fisher Scientific (1 mentions)
Axiovision software v4, by Zeiss (1 mentions)
Eclipse te2000 u microscope, by Nikon (1 mentions)
Depex, by Serva Electrophoresis (1 mentions)
Application suite version 4, by Leica (1 mentions)
Dmi8 microscope, by Leica (1 mentions)
Roti histokit, by Carl Roth (1 mentions)
Entellan, by Merck Group (1 mentions)
Euparal, by Carl Roth (1 mentions)
Discovery v8 stereomicroscope, by Zeiss (1 mentions)
Mayer s hemalum solution, by Carl Roth (1 mentions)
Axiovert s100 microscope, by Zeiss (1 mentions)
Axiocam mrm camera, by Zeiss (1 mentions)
Cytoseal, by Thermo Fisher Scientific (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
Vectashield mounting medium for fluorescence, by Vector Laboratories (1 mentions)
Rm2165, by Leica (1 mentions)
Mayer s hematoxylin, by Carl Roth (1 mentions)
9 protocols using eosin solution
1
Histological Analysis of Patient-Derived OA Chondrocytes
2
Histological Assessment of Brain Morphology
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To assess the general brain morphology, haematoxylin and eosin (HE) stain was used as described in [11 (link)] for placental analyses. In short, sections were deparaffinized, rehydrated, 5 min incubated with haematoxylin solution (Thermo-Scientific, Pittsburgh, USA), and shortly washed in tap water and ethanol (70%)/HCl (0.5%) solution for differentiation, followed by 5 min bluing in flowing tap water. Afterwards, sections were incubated for 3 min in eosin solution (#7089.1, Carl Roth, Karlsruhe, Germany) and washed several times in 96% ethanol for differentiation, followed by a standard dehydration procedure and mounting in xylene mountant. With HE stain, nuclei are displayed in blue and cytoplasm is displayed in red. HE stain was used for morphometric analyses of brain volume and brain region areas (about 14 images per sample) as well as nucleus counting (nine images per sample).
3
Evaluating Tendon Sheet Alignment
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4 Y-TSPC (n = 4 donors) and 6 A-TSPC (n = 6 donors), the sheets (1 sheet/donor) were placed in Hematoxylin solution for 3 min, rinsed with 0.1% HCl in PBS for dedifferentiation, washed in tap water for 5 min, immersed in Eosin solution for 3 min (both from Carl Roth, Karlsruhe, Baden-Württemberg, Germany), rinsed with distilled water for 30 s and covered with Depex (Serva, Heidelberg, Germany). H&E staining images were made with Nikon digital sight DS-U camera mounted at Nikon eclipse TE2000-U microscope (Nikon, Tokyo, Japan) and used for analysis of nuclear angular deviation. Since healthy resident cells in tendon tissue are well aligned to the tensile axis (muscle to bone), this parameter is useful to assess the quality of the 3D sheet organization, and the lesser the deviation the better the alignment. The following algorithm was implemented: (1) 9 images at x20 magnification were taken randomly from each cell sheet; (2) 9 randomly chosen nuclei per image were assessed (81 nuclei/sheet); (3) the angles between the longitudinal axis of the cell sheet and the long axis of the nuclei were determined with the “angular” tool of the AxioVision software v 4.8 (Carl Zeiss, Jena, Germany); (4) 324 and 486 angles for Y- and A-TSPC cohorts were analyzed, respectively; (5) the distribution of nuclear angle deviation for each cell type was generated with GraphPad Prism.
4
Hematoxylin and Eosin Staining of RCC-EW Cells
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Prior to staining of RCC-EW with hematoxylin and eosin (H&E), 3.2×104 cells per well of a 12-well plate were seeded on cover slips and treated for 96 hours with adenovirus. Afterward, the cells were washed with PBS, fixed with methanol:acetone (1:1) for 10 minutes at −20°C. For H&E staining, the fixed cells were incubated with hematoxylin solution modified according to Gill II (Carl Roth) for 15 minutes, rinsed with tap water, incubated with eosin solution (Carl Roth, 75 mg eosin in acidic 75% ethanol) for 1 minute, and dehydrated in ascending ethanol concentrations (from 80% to 100%). After mounting the cover slips on slides using Roti Histokit (Carl Roth), staining was visualized with a Leica DMi8 microscope using a MC170HD camera and the Leica Application Suite version 4.4.
5
Histopathological Characterization of Healthy and Inflamed Third Molars
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The healthy and inflamed human third molars were histopathologically characterized by hematoxylin and eosin (H&E) staining, as previously described [45 (link)]. The sections were first dried at room temperature for two hours. Subsequently, the sections were washed in distilled water for 5 min and treated with a hematoxylin solution (Carl-Roth GmbH, Karlsruhe, Germany) for 10 min. The sections were washed with slightly warmer running water for 10 min. Following additional washing of the sections with distilled water for 2 min, the sections were treated with eosin solution (Carl-Roth GmbH) for 90 s. The sections were dehydrated in an ascending ethanol series (90%, 95%, 2 × 100% ethanol) for 3 min each. Subsequently, the sections were treated with xylene-I and xylene-II (Carl-Roth GmbH) for 4 min each and coverslipped with Entellan (Merck Millipore, Darmstadt, Germany).
6
Corneal Paraffin Section Staining
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After an initial brief rinse in dH2O, the corneal paraffin sections were stained for 3 min with Mayer’s hemalum solution (Carl Roth, GmbH + Co. KG, Karlsruhe, Germany). The sections were then briefly washed again in dH2O and then blued for 20 min under running tap water. After another washing step in dH2O, the samples were stained with eosin solution (Carl Roth, GmbH + Co. KG, Karlsruhe, Germany) for 30 s, washed in dH2O and immersed for a short time in solutions of ascending alcohol series (70, 80, 90, 96%). Finally, the sections were covered with Euparal (Carl Roth, GmbH + Co. KG, Karlsruhe, Germany) and left to dry for at least 1 h. Tissue sections were analysed with the Discovery.V8 stereomicroscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) and histological overviews were created using the mosaic tool.
7
Cryosectioning and Histological Staining
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The cell sheets were fixed in 4% paraformaldehyde/PBS, cryoprotected with sucrose, embedded in tissue freezing medium (Jung, Leica, Nussloch, Baden-Württemberg, Germany) and cryosectioned at 10 μm (Microm HM500 OM, Fisher, Walldorf, Baden-Württemberg, Germany). Sections were stored at −20 °C until use. Before staining, sections were equilibrated to room temperature and hydrated with PBS (3 × 5 min) and rinsed with deionized water. For Haematoxylin and Eosin (H&E), sections were placed in a ready-to-use Hematoxylin solution (Carl Roth, Karlsruhe, Baden-Württemberg, Germany) for 10 min, rinsed with deionized water for 5 min and washed in tap water for 7 min. Next, sections were immersed in a ready-to-use Eosin solution (Carl Roth, Karlsruhe, Baden-Württemberg, Germany) for 10 min, rinsed with distilled water for 30 s, and mounted. Alexa Flour 546-labelled phalloidin (Invitrogen, Karlsruhe, Baden-Württemberg, Germany) was applied according to the manufacturer’s recommendation, as described previously [6 (link)]. DAPI was used for nuclear counterstaining. Cell sheets were imaged with the AxioCamMRm camera on the AxiovertS100 microscope (Carl Zeiss, Jena, Thüringen, Germany). For quantification of the total actin amount, fluorescence signals were quantified using the algorithm described above.
8
Histological Analysis of Mouse Brain
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Histological stainings with haematoxylin and eosin (HE) were performed with samples obtained at 28 dpi. Brain slices were prepared as aforementioned. The desired plane containing the striatum as a region of interest was placed in a cryomold, embedded in Tissue Tek O.C.T. Compound (# 4583, Sakura Finetek, Netherlands) and fresh frozen on dry ice. Samples were stored at −80 °C until further processing. 7 μm thick cryosections were cut and stored at −20 °C until staining.
Sections were post-fixed with 4% paraformaldehyde (PFA) for 10 min and briefly washed with distilled water before incubation in haemalaun solution (#T865.2, Carl Roth, Germany) for 10 min. After washing with warm, flowing tap water for 15 min, sections were again washed with distilled water for 2 min before incubation in eosin solution (#X883.2, Carl Roth, Germany) containing acetic acid for 45 s. Sections were again thoroughly washed with tap water before undergoing treatment with ascending ethanol concentrations (70%–96% - 100%). Subsequently, sections were washed in xylene (2 × 3 min) and mounted with Cytoseal (# 8312-4, ThermoFisher Scientific, USA) to prevent bleaching.
Sections were post-fixed with 4% paraformaldehyde (PFA) for 10 min and briefly washed with distilled water before incubation in haemalaun solution (#T865.2, Carl Roth, Germany) for 10 min. After washing with warm, flowing tap water for 15 min, sections were again washed with distilled water for 2 min before incubation in eosin solution (#X883.2, Carl Roth, Germany) containing acetic acid for 45 s. Sections were again thoroughly washed with tap water before undergoing treatment with ascending ethanol concentrations (70%–96% - 100%). Subsequently, sections were washed in xylene (2 × 3 min) and mounted with Cytoseal (# 8312-4, ThermoFisher Scientific, USA) to prevent bleaching.
9
Histological Examination of Decellularized Samples
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Histological examination of samples underlying decellularization and of control samples was performed using hematoxylineosin (HE), Elastica-van-Gieson (EvG) and 4′,6-diamidin-2phenylindol (DAPI) staining.
For HE staining, paraffin-embedded tissue was cut into 5 µm sections using a rotary microtome (RM 2165, Leica, Wetzlar, Germany), transferred to slides and dewaxed, followed by 5 min incubation in Mayer's hematoxylin (Roth, Karlsruhe, Germany), 5 min wash in running tap water and 3 min incubation in eosin solution (0.5%, Roth). EvG staining was done by 1 h incubation in Weigert's resorcin-fuchsin solution (Roth), iron hematoxylin (Roth) for 3 min and van-Gieson-solution (Hollborn, Leipzig, Germany) for 10 min. For DAPI staining, paraffin-embedded samples were mounted with Vectashield Mounting Medium for fluorescence (Vector Laboratories, Inc., CA, USA). After staining, slides were imaged by light and fluorescence microscopy (AxioVision, Zeiss, Jena, Germany) using 100 x, 200 x and 400 x objectives.
For HE staining, paraffin-embedded tissue was cut into 5 µm sections using a rotary microtome (RM 2165, Leica, Wetzlar, Germany), transferred to slides and dewaxed, followed by 5 min incubation in Mayer's hematoxylin (Roth, Karlsruhe, Germany), 5 min wash in running tap water and 3 min incubation in eosin solution (0.5%, Roth). EvG staining was done by 1 h incubation in Weigert's resorcin-fuchsin solution (Roth), iron hematoxylin (Roth) for 3 min and van-Gieson-solution (Hollborn, Leipzig, Germany) for 10 min. For DAPI staining, paraffin-embedded samples were mounted with Vectashield Mounting Medium for fluorescence (Vector Laboratories, Inc., CA, USA). After staining, slides were imaged by light and fluorescence microscopy (AxioVision, Zeiss, Jena, Germany) using 100 x, 200 x and 400 x objectives.
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