After overnight incubation at 60°C and application of a series of 2 xylene solutions for 30 min each, 5 mm paraffin cross-sections were deparaffinized using a Rotary microtome (RM 2135, Leica) assistant. The cross-sections were then treated with an alcohol series with decreasing concentrations from 95% to 60% and then soaked in flowing water for 5 min to complete the rehydration. The cross-sections were stained following standard protocols for hematoxylin and eosin (HE) and periodic acid–Schiff (PAS) staining. In the same manner, after 5 min of soaking under flowing water and treatment with alcohol series of 80% and 95% alcohol solutions, the cross-sections were air-dried. The aim of this procedure was to make each of the samples pellucid within 30 min of being treated with two entellan-xylene mixtures (UN 1866, Merck, Darmstadt, Germany). The cross-sections were then analyzed by light microscope dispersion. A pathologist evaluated the testicular tissues using standard light microscopy. This examination was completed in a random order and a blinded fashion. The histologic sections were graded for testicular injury and spermatogenesis using the Johnsen score (JS). A minimum of 50 tubules were evaluated, and each tubule was given a score from 1 to 10.
Rotary microtome
Manufactured by Leica camera
Sourced in Germany, United States, United Kingdom
The Rotary Microtome is a precision instrument used for cutting thin, uniform sections of specimens for microscopic examination. It features a rotational mechanism that advances the specimen through a stationary knife, allowing for the creation of very thin tissue sections.
Automatically generated - may contain errors
Lab products found in correlation
Harris hematoxylin solution, by Merck Group (7 mentions)
Eosin y solution, by Merck Group (5 mentions)
Slide warmer, by Thermo Fisher Scientific (4 mentions)
Cresyl violet, by Merck Group (4 mentions)
Eclipse e600 research microscope, by Nikon (4 mentions)
Virtuoso v 5, by Roche (4 mentions)
Embedding system, by Leica camera (3 mentions)
Embedding center, by Leica camera (3 mentions)
Paraffin embedding center, by Leica camera (3 mentions)
Eclipse 80i, by Nikon (3 mentions)
Ventana benchmark ultra system, by Roche (3 mentions)
Formalin, by Merck Group (3 mentions)
Paraffin, by Merck Group (3 mentions)
Leica dm4000b, by Leica camera (2 mentions)
Sabc kit, by Boster Bio (2 mentions)
Dab kit, by Boster Bio (2 mentions)
Anti pak2, by Abcam (2 mentions)
Anti ki67, by Abcam (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Permount mounting medium, by Thermo Fisher Scientific (2 mentions)
209 protocols using rotary microtome
1
Histological Evaluation of Testicular Tissue
2
Histological Analysis of Mouse Embryos
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Embryos were dissected from mice immediately after euthanasia, fixed in 4% paraformaldehyde (PFA) for up to 24 h, stored in 70% ethanol and embedded in paraffin. Then 5 μm-thick sections were prepared using rotary microtome (Leica) and mounted on glass slides.
After deparaffinization, sections were stained with hematoxylin and eosin (H&E) for histological analysis.
After deparaffinization, sections were stained with hematoxylin and eosin (H&E) for histological analysis.
3
Kidney preservation and assessment
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The rats in all groups were sacri ced by overdoses of sodium pentobarbital. Except those in the three burn groups (sacri ced at 6 h, 12 h, 24 h post burn), the animals in the other groups were sacri ced at 48 h post insults. Both kidneys were dissected after cardiac perfusion with phosphate-buffered saline (PBS) (pH = 7.2) and were maintained in 4% paraformaldehyde at 4°C or in a -80°C freezer for subsequent assessment. The frozen or para n-embedded kidney samples were sectioned by using a Cryostat Microtome or a Rotary Microtome (Leica, Solms, Germany) for further staining.
4
Histological Analysis of Lung Tissues
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Isolated lung tissues were fixed using 4% paraformaldehyde at room temperature for 24 h and then embedded in paraffin, and were cut into 4 μm sections using a rotary microtome (Leica, Mannheim, Germany). The dried slices were soaked in xylene, dewaxed for 10 min, rehydrated, then performed hematoxylin rinse for 5 min and washed, followed by differentiation with 1% hydrochloric acid alcohol. Then slides were stained with eosin for 30 s, dehydrated with gradient alcohol, soaked in xylene three times. Finally, mounted slides with neutral gum and analyzed under an optical microscope (DP73; Olympus Corporation, Tokyo, Japan). The process of taking images was conducted by an assessor blind to treatment allocation.
5
Immunohistochemical Analysis of Mouse Tumors
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Specimens from necropsied mice were fixed in 10% neutral buffered formalin (Surgipath Leica, Buffalo Grove, IL, USA) and embedded in paraffin. Five-micron sections were sliced using a rotary microtome (Leica). SelecTech hematoxylin and eosin reagents (Surgipath, Buffalo Grove, IL, USA) were used for histological staining. For immunohistochemistry, antigen retrieval was performed with either sodium citrate, pH 6.0, or EDTA, pH 9.0 (Leica, Buffalo Grove, IL, USA). Primary antibodies were against MYC (Abcam, Cambridge, MA, USA), MPO (Thermo Fisher Scientific, Waltham, MA, USA) or F4/80, CD86, CD3, CD4, or CD8 (all from Cell Signaling Technology, Danvers, MA, USA). A Bond-Max Immunostainer and Polymer Refine Detection reagents (Leica, Buffalo Grove, IL, USA) were used for staining. Multiple fields of view of each tumor section were imaged across three representative mice from each treatment group on a Keyence BZ-X800 microscope. Antibody detection was quantified using Keyence BZ-X800 analysis software. Results are reported as mean ± SD. To compare each mean with every other mean, a one-way ANOVA followed by post hoc Tukey’s test for multiple comparison was used to analyze statistical significance between treatment groups. (p < 0.05 [*], p < 0.01 [**], p < 0.001 [***], p < 0.0001 [****]).
6
Histological Analysis of Rice Lamina Joints
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GUS activity assays were performed in T0 and T1 transgenic plants. Briefly, tissues were incubated in staining buffer (50 mM sodium phosphate, pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 1 mg ml À1 X-Gluc, 100 lg ml À1 chloramphenicol, 0.5 mM potassium ferricyanide, 1 mM potassium ferrocyanide, and 20% methanol) at 37°C and then cleaned in a concentration gradient of ethanol. The stained tissues were observed and photographed with a stereomicroscope (Leica DFC495).
For our histological analysis, the lamina joints of 10-dayold rice plants were dissected and fixed in iced FAA solution (50% ethanol, 5% acetic acid, and 3.7% formaldehyde). After shaking at 4°C overnight, the samples were dehydrated and stained with eosin in a graded series of ethanol solutions from 50 to 100%. The dehydrated samples were then embedded using paraffin or resin and cut into 5-10lm sections with a rotary microtome (Leica). The sections from each sample were observed and photographed under a light microscope (Leica DFC495). The abaxial and adaxial sclerenchyma cells in each section were counted and observed under a light microscope.
For our histological analysis, the lamina joints of 10-dayold rice plants were dissected and fixed in iced FAA solution (50% ethanol, 5% acetic acid, and 3.7% formaldehyde). After shaking at 4°C overnight, the samples were dehydrated and stained with eosin in a graded series of ethanol solutions from 50 to 100%. The dehydrated samples were then embedded using paraffin or resin and cut into 5-10lm sections with a rotary microtome (Leica). The sections from each sample were observed and photographed under a light microscope (Leica DFC495). The abaxial and adaxial sclerenchyma cells in each section were counted and observed under a light microscope.
7
Immunohistochemistry Protocol for Tissue Sections
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Immunohistochemistry was performed as described before (Grazul-Bilska et al. 2006 (link), 2010 , 2011) . Briefly, all tissues were sectioned using a Leica rotary microtome (Leica RM 2255) and mounted on poly-l-lysine-treated slides (Thermo Fisher Scientific). Tissue sections were deparaffinized in xylene and rehydrated in decreasing concentrations of alcohol in water and then washed in distilled water. Tissue sections underwent antigen recovery (2100 Retriever, Prestige Medical, Lancashire, England) for 20 min in a 10 mM sodium citrate buffer with 0.05% Tween (pH 6). Slides with tissue sections were allowed to cool at room temperature for 20 min, washed and then blocked with 10% normal goat serum for 20 min at room temperature followed by incubation with a specific primary antibody and then with a secondary antibody. Table 1 presents fixatives used, tissue thickness and source and dilution of primary and secondary antibodies for all antigens immunodetected. Tissue sections were cover-slipped using Prolonged Gold with DAPI (Life Technologies).
8
Histological Evaluation of Implanted Polyethylene
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The rats were anesthetized with 90 mg/kg ketamine hydrochloride and 5 mg/kg xylazine hydrochloride to evaluate the results on 1, 2, and 4 weeks after material implantation. Later, the blood samples collected by intracardiac method was delivered to the biochemistry laboratory with heparinized tubes. Rats were killed by intracardiac exsanguination under anesthesia.
The surrounding connective tissue which had been in contact with the polyethylene tubes was removed. At the same time, the liver and left kidney were removed. The tissue samples from the liver, kidney and connective tissue were placed in 10% formalin for 48 h, then embedded in paraffin blocks. Slices 5 µmin thickness were cut with a Leica Rotary microtome, and the preparates were stained with hematoxylin and eosin and Mallory Trichrome for light microscope evaluation.
The surrounding connective tissue which had been in contact with the polyethylene tubes was removed. At the same time, the liver and left kidney were removed. The tissue samples from the liver, kidney and connective tissue were placed in 10% formalin for 48 h, then embedded in paraffin blocks. Slices 5 µmin thickness were cut with a Leica Rotary microtome, and the preparates were stained with hematoxylin and eosin and Mallory Trichrome for light microscope evaluation.
9
Quantitative TNF-alpha Expression Analysis
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The left cerebral hemisphere that had been inserted into the fixation fluid was dehydrated using gradient alcohol and xylene, then paraffinized and cut as thick as 5 um using a rotary microtome (Leica). Next, tissue was placed on coated-object glass. Then, rehydration was carried out on the tissue using xylene and alcohol with a concentration of 96%, 90%, 80%, and 70% and rinsed with tap water. The next stage, retrieval antigen was carried out by the heatinduced epitope retrieval method, where the slides were put into a citrate buffer solution, then heated at a temperature of 95°C for 60 min. Then, tumor necrosis factor (TNF)-alpha 1:700 (cloud clone) antibody was stained, followed by overnight incubation at 4°C. The next stage was to paint with a secondary antibody, biotinylated-horseradish peroxidase, and incubation for 1 h at room temperature. Next, chromogen was added to the slide. Furthermore, the dehydration process was again carried out using concentrated alcohol and xylene. The next step was mounting and evaluating the TNF-alpha expression using ImageJ Software so that the percentage of TNF-alpha expression was obtained.
10
Histomorphology Analysis of Intestinal Tissue
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The xed tissues were dehydrated and xed with para n in a 2T-12M tissue processor (XiaoganYaguang Medical Electronic Technology Co., Ltd., Xiaogan, P. R. China), and then embedded in para n blocks using an embedding system (Leica, Germany). The blocks were then sliced into 7 µm-thick sections using a rotary microtome (Leica, Germany) and stained with Haematoxylin & Eosin (HE). The crypt depth (Cd, from the basis of the villus to the submucosa), villus height (Vh, from the tip of the villus to the crypt) and the villus height to crypt depth (Vh/Cd) ratio were evaluated [22] . Histomorphology analyses were performed on 6 well-oriented and intact villi and 6 crypts chosen from duodenum, jejunum, ileum and cecum. Slices were photographed and measured using an EVOS™ M5000 Imaging System (Thermo Fisher Scienti c, America).
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