Tissue blocks were paraffin-embedded and cut on a microtome into 3–5 µm thick sections. For histological characterization of the MRI features, the following antibodies were used: CD68 (DAKO, clone PG-M1, 1:100), and GFAP (Dako, GA52461-2, 1:100). For immunohistochemistry, slides were incubated with PBS before and blocking in PBS/10% bovine serum albumin for 1 hour at room temperature. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide for 20 min. Slides were incubated with primary antibodies overnight at 4°C. Secondary biotinylated antibody was applied for 1 hour at room temperature followed by the ABC complex reagent (VectorLabs) for one hour. The colour reaction was carried out with ‘ImpactDAB’ (VectorLabs). All counterstainings were done with Haematoxylin. In addition, Luxol Fast Blue staining was used to assess myelination, Prussian Blue for hemosiderin, and Elastica van Gieson staining to highlight elastic lamina in vessel walls. Subsequently, slides were dehydrated in ethanol/xylol and mounted using ‘Entellan’ (Merck Millipore). Slides were scanned using a Hamamatsu slide scanner (Hamamatsu Photonics). Corresponding microphotographs of regions of interest were taken using NDP.view2 viewing software.
Hamamatsu slide scanner
Manufactured by Hamamatsu Photonics
Sourced in Japan
The Hamamatsu slide scanner is a device designed to digitize glass microscope slides. It captures high-resolution images of the slides, preserving the details and information contained within them.
Automatically generated - may contain errors
Lab products found in correlation
Apoptag peroxidase in situ apoptosis detection kit, by Merck Group (2 mentions)
Impact dab, by Vector Laboratories (1 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (1 mentions)
Entellan, by Merck Group (1 mentions)
Abc complex reagent, by Vector Laboratories (1 mentions)
Z series coulter counter, by Beckman Coulter (1 mentions)
Giemsa, by Merck Group (1 mentions)
3 amino 9 ethylcarbazole, by Merck Group (1 mentions)
4 protocols using hamamatsu slide scanner
1
Histological Characterization of MRI Features
2
Neutrophil Influx and Cytokine Analysis
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The number of cells in the peritoneal lavage fluid was measured using a Z Series coulter counter (Beckman Coulter, Brea, CA, USA). After counting, cells were diluted in 5.0 × 105 cells mL−1, and 100 µL was used to prepare cytospins. The remaining solution was spinned down at 3000 × g and stored at −80 °C for cytokine measurements. The cytospin slides were stained with Giemsa (Merck Millipore) for 30 min at room temperature. The stained slides were scanned at a magnification of 20× using a Hamamatsu slide scanner (Hamamatsu Photonics), and neutrophil influx in peritoneum was determined by counting the number of neutrophils within 150 immune cells.[7 ] The total number of neutrophils was calculated using the total cell count of the peritoneal lavage fluid. Cytokines (TNF‐α, IL‐6 and IL‐10, Gro‐a, and MCP‐1) from the peritoneal fluid were determined with ELISA (R&D systems) according to manufacturer's instructions.
3
Quantification of Intestinal Apoptosis
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Ileal samples from mice were fixed in 10% formalin in PBS and embedded in paraffin. Paraffin blocks were cut in 4 µm sections. The tissue sections were analyzed for apoptosis in crypts using a TUNEL assay. The TUNEL assay was performed using the ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Merck Millipore, Billerica, MA, USA) according to the instructions of the manufacturer. Hematoxylin was used for counterstaining the slides. As peroxidase substrate, an incubation step of 15 min with 3-amino-9-ethylcarbazole (AEC) (5% AEC stock; 95% 0.05 M acetate buffer pH 4.9; 0.1% of 30% v/v H2O2) (Merck Millipore, Billerica, MA, USA) was used. The stained slides were scanned with a Hamamatsu slide scanner (Hamamatsu Photonics, Hamamatsu City, Japan), and analysis was performed using NDP.view2 Software. TUNEL positive cells were measured in 10 sequential crypts in the ileum per mouse.
4
Histological and Apoptotic Evaluation of Ileum
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Ileum was cut in pieces of 0.3 mm and they were fixed in 4% paraformaldehyde in PBS and embedded in paraffin. Paraffin sections of 4 µm were cut and hematoxylin and eosin (H&E) staining was performed on the slides. The ileum sections were also stained for apoptosis using the TUNEL assay. The TUNEL assay was performed according to manufacturer's instructions of the ApopTag Peroxidase In Situ Apoptosis Detection Kit (Merck Millipore, Billerica, MA, USA). As peroxidase substrate, 3‐amino‐9‐ethylcarbazole (AEC) (5% AEC stock; 95% 0.05 M acetate buffer pH 4.9; 0.1% of 30% v/v H2O2 (Merck)) was used and incubated for 10 min. Hematoxylin was used as counterstain. The stained slides were scanned at a magnification of 40× using a Hamamatsu slide scanner (Hamamatsu Photonics, Hamamatsu, Japan). Histopathological scoring (ranging from 0 to 12) was performed by assessing epithelial‐, villus‐, and crypt damage, and stroma retraction on H&E stained slides by two individuals as previously described.[7 ] Villus height and villus thickness were determined from 10 consecutive villi structures of three different ileum segments. The villus height was measured from the base of the villus to the top. Villus thickness was measured at the base of a villus. Apoptotic cells were measured in 10 sequential crypts in the ileum per mouse.
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