The small intestines of sacrificed mice were removed, washed and the ileum parts were collected and fixed overnight in 4% PFA solution (paraformaldehyde). Afterward, the tissues were dehydrated in increasing concentrations of alcohol, cleared in xylene, and embedded in paraffin wax using conventional methods (Alturkistani et al., 2016 (link)). Then histological slices of 4μm were prepared from paraffin blocks using a rotation microtome (Leica RM2255 Automated Microtome). Processing, paraffin embedding, and histological slices were performed by The University of Ottawa histology core facility.
Rm2255 automated microtome
Manufactured by Leica
Sourced in Germany
The RM2255 Automated Microtome is a laboratory equipment manufactured by Leica. It is designed for the precise sectioning of specimens, producing thin sections for microscopic analysis. The RM2255 features automated operation and precision controls to ensure consistent and accurate sample preparation.
Automatically generated - may contain errors
4 protocols using rm2255 automated microtome
1
Histological Preparation of Mouse Ileum
2
Histological Processing of Mouse Ileum
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The ileum tissues of mice were removed, washed with ice-cold PBS, and small sections of the ileum were collected and fixed in a 4% paraformaldehyde solution for 48 h. Subsequently, fixed tissues were dehydrated in increasing alcohol concentrations, cleared in xylene, and embedded in paraffin using conventional methods. Histological sections of 4 µm were prepared from paraffin blocks using a rotational microtome (Leica RM2255 Automated Microtome). The processing, embedding, and preparation of histological sections were performed by the University of Ottawa Histology Core Facility.
3
Immunohistochemical Detection of PD-L1 in Tissue Samples
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Slides were stained for routine diagnostics, over a period of several years. Formalin‐fixed paraffin‐embedded blocks were cut into 3‐µm sections with a Leica RM2255 Automated Microtome (Leica Biosystems B.V., Amsterdam, the Netherlands). Sections were placed on microscope slides and dried at either 60°C for 30 min to 16 h, or at 37°C for 72 h. After being dried, the slides were deparaffinised, and antigen retrieval was performed in citrate buffer (Target Retrieval Solution, pH 6) for 40 min. Immunohistochemistry (IHC) was performed according to a laboratory‐developed test protocol. Slides were stained with the Dako Omnis immunostainer and Dako EnVision Flex+ reagents and 1:20 dilution of PD‐L1 clone 22C3 (Dako Omnis, Dako Agilent Technologies, Leuven, Belgium). The IHC slides were then counterstained with haematoxylin, and coverslips were applied. Tonsil and placental tissue were used as positive controls for PD‐L1 expression.
4
Histomorphometric Analysis of Murine Bone
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Femurs from 3‐ and 7‐month‐old male and female mice were isolated and fixed in 4% paraformaldehyde solution and decalcified using 10% EDTA for 2 weeks. Samples were then dehydrated using a graded ethanol series and were paraffin‐embedded. Coronal sections (5 μm) were obtained using the Leica RM 2255 automated microtome (Leica Biosystems, Wetzlar, Germany), and stained for alkaline phosphatase (ALP) or tartrate‐resistant acid phosphatase (TRAP), for osteoblast and osteoclast visualization, respectively. High‐resolution slide scans were acquired using the Aperio At Turbo Digital Pathology Scanner (Leica Biosystems). For each randomly numbered sample, 3 to 5 sections were analyzed in full for bone cell numbers, at 4× magnification, using the ImageJ software (NIH). Undecalcified bones were embedded in methyl methacrylate (MMA), and 1‐μm sections were cut on an ultramicrotome. These sections were stained with the Goldner trichrome method as previously described.(22 ) All histological analyses were conducted as per the recommendations of the American Society for Bone and Mineral Research Histomorphometry Nomenclature Committee.(23 )
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