Immediately after the final behavioral test (PN40), rats were sacrificed by decapitation using a guillotine. Brains were quickly removed, immediately fixed with 4% paraformaldehyde in PBS for 24 h, immersing in 30% sucrose for 5 days, embedded in OCT at −70°C and cut into 20-μm thick coronal sections through the striatum using a cryostat. Free-floating sections were stained for the dopaminergic marker TH. In brief, sections were incubated in 0.3% hydrogen peroxide for 6 min at RT to quench endogenous peroxidase activity, permeabilized by emersion in 0.1% PBST for 10 min (3 times), blocked in PBST supplemented with 1% bovine serum albumin for 2 h and incubated with anti-TH (1:1000) overnight at 4°C. The next day, sections were incubated with a biotinylated secondary antibody for 1 h at RT and stained using a 3-3-diaminobenzidine tetrahydrochloride (DAB) substrate kit (BD Bioscience). Sections were dried, mounted on slides using Entellan and imaged using a ScanScope CS scanner (Aperio Technologies, Vista, CA) and ImageScope software (Aperio).
Scanscope cs scanner
Manufactured by Leica
Sourced in Germany
The ScanScope CS is a high-resolution slide scanner from Leica Biosystems. It digitizes pathology slides at up to 40x magnification, capturing images with a resolution of up to 0.25 micrometers per pixel. The scanner is designed for use in research and clinical laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Imagescope software, by Leica (3 mentions)
Tissue plus o c t, by Thermo Fisher Scientific (2 mentions)
Axio imager m2, by Zeiss (2 mentions)
In situ cell death detection kit, by Roche (2 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (1 mentions)
Anti p erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Anti ki67, by Agilent Technologies (1 mentions)
Evos xl core cell imaging system, by Thermo Fisher Scientific (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
6 protocols using scanscope cs scanner
1
Quantitative Analysis of Dopaminergic Neurons
2
Immunostaining of Pancreatic Tissue for Cell Type Analysis
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For immunostaining, WT and MafAS64F/+ pancreata were fixed in 4% (v/v) paraformaldehyde, embedded in either Tissue-Plus O.C.T. (Thermo Fisher Scientific) or paraffin wax, and sectioned to 6 mm thickness. Immunofluorescent images were obtained using a Zeiss Axio Imager M2 widefield microscope with ApoTome. Immunofluorescence staining was performed as previously described with the antibodies listed in Key resources table . Islet β- and α- cell areas were determined as described previously (Cyphert et al., 2019 (link)). Briefly, pancreatic sections taken every 50 μm (n = 3-4 animals per genotype) were scanned using a ScanScope CS scanner (Aperio Technologies, Vista, CA). Images from each experiment were processed with ImageScope Software (Aperio Technologies, Vista, CA). Islet β- and α-cell areas were calculated as the ratio of the insulin- or glucagon-positive area to total pancreas area (eosin stained). The TUNEL assay was performed using an in situ cell death detection kit (Roche, #11684795910). In EndoC-βH2 cells, immunostaining for LaminB1, 53BP1, and p21 were also performed (see Key resources table ).
3
Immunostaining of Pancreatic Tissue for Cell Type Analysis
4
Quantifying Pancreatic β-Cell Mass
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Sections were cut serially at 5 μm and placed on slides pre-treated with Sta-On (Leica Biosystems, Lincolnshire, IL). Six to seven slides per animal (separated by at least 250 μm and sampling the entire pancreas) were immunolabeled for insulin, visualized via the DAB Peroxidase Substrate Kit (Vector Laboratories, Burlingame, CA), and counterstained with eosin. Quantification of β-cell mass was calculated as described previously [22 (link)]. In brief, one pancreatic section per slide was scanned using a ScanScope CS scanner, and the images were processed identically with the ImageScope software (Aperio Technologies, Vista, CA). β-cell mass was measured by determining the ratio of insulin + area to the total pancreatic area of all the scanned sections per animal multiplied by the wet weight of the pancreas.
5
Immunohistochemical Detection of Cleaved Caspase 3
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Livers were fixed in 4% PFA, dehydrated in successive baths (30%, 70%, 95%, and 100% ethanol and toluene), and paraffin-embedded. Immunohistochemistry was performed to detect cleaved caspase 3 using a Ventana Discovery XT autostainer on 4 μm-thick sections. Slides were deparaffinized with EZPrep buffer and epitopes unmasked in CC1 EDTA buffer. Sections were incubated 40 min with anti-cleaved caspase 3 antibody (1/1000). Secondary antibody (Omnimap Rabbit Ventana) was incubated 16 min. After washes, staining was performed with DAB and sections were counterstained with Hematoxylin. Whole slide images were digitized at 20 × using the ScanScope CS scanner (Leica Biosystems, Nussloch, Germany).
6
Quantifying Liver Cyst Formation
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Sections of the mouse livers were suspended in formalin (10% phosphate-buffered formaldehyde) for 1–2 days and were subsequently dehydrated and embedded in paraffin blocks. Paraffin blocks were prepared into slides stained by hematoxylin and eosin (H&E), anti-Ki-67 (1∶100, monoclonal rat anti-mouse, #M7249, Dako, Carpinteria, CA), anti-HSP90α (1∶800, monoclonal rabbit, #NB120-2928, Novus Biologicals, Littleton, CO), anti-pERK1/2Thr202/Tyr204 (polyclonal, rabbit, #9101, Cell Signaling, Danvers, MA), or cleaved caspase-3 (1∶200, polyclonal, #9661 Cell Signaling, Danvers, MA) using standard procedures described previously [20] (link). All slides containing immunohistochemical staining were imaged using a ScanScope CS scanner (Aperio, Leica Biosystems, Buffalo Grove, IL). To quantify cystic indices, the ImageJ program was used to generate computerized grids with an area per point of 5000 µm2 and to superimpose them upon images of H&E-stained liver sections scanned at 10x magnification using an EVOS XL Core Cell Imaging System (Life Technologies, Grand Island, NY). Cystic indices were calculated as a percentage of grid intersections that crossed cysts, with ∼1450 grid intersections analyzed for per mouse. Clear areas with diameters >40 µm were defined as cysts; those with diameters >20 µm defined as dilated tubules.
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