After sacrifice, the colon was removed and its length measured. The colon was flushed with ice-cold PBS, cut open longitudinally and rolled up from distal to proximal to obtain a so-called “swiss-role” [22 ], which was then fixed in 4% PFA (Carl Roth, Karlsruhe, Germany) overnight at room temperature. After dehydration and embedding in paraffin, 2 μm sections were prepared and stained with hematoxylin and eosin (H&E). Photomicrographs were acquired using a Leica Axio Scope A1 microscope (Wetzlar, Germany). Histopathological evaluation of the entire section was performed in a blinded manner by determining crypt distortion, inflammation and loss of goblet cells. Based on these criteria an inflammatory score from 0 to 3 (0 = none, 1 = mild, 2 = moderate, 3 = severe) was assigned, multiplied with the percentage of the affected tissue area, and depicted on a scale from 0 to 10.
Axio scope a1 microscope
Manufactured by Leica
Sourced in Germany
The Axio Scope A1 is a microscope designed for routine and advanced microscopy applications. It features a stable and ergonomic stand, a wide range of objectives, and a variety of illumination options to accommodate different sample types and magnification requirements. The Axio Scope A1 provides high-quality imaging capabilities to support a variety of research and analysis tasks.
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6 protocols using axio scope a1 microscope
1
Histological Evaluation of Colonic Inflammation
2
Immunohistochemical Analysis of CD3 and CD68 in Tissue Sections
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For immunohistochemical stainings, 2 μm tissue sections were incubated in EnVision Flex Target Retrival Solution, Low pH (Dako Agilent Technologies, Santa Clara, CA). This step was followed by incubation with primary antibodies recognizing CD3 (1:2000; Santa Cruz Biotechnology) or CD68 (1:200; Abcam, Cambridge, UK) for 30 minutes at room temperature. Polymeric secondary antibodies coupled to HRP (mmPRESS HRP Polymer Detection Kit; Vector Laboratories, Burlingame, CA) and DAB (Dako Agilent Technologies) were employed to visualize the sites of immunoreactivity. Counterstaining was done with hematoxylin. Photomicrographs were acquired using a Leica Axio Scope A1 microscope and quantified after taking ten pictures per section at 20x magnification. To this end, pictures were processed using ImageJ (https://imagej.nih.gov/ij/ ) either by counting individual cells (CD3) or by measuring the stained tissue areas (CD68).
3
Quantifying Cholinergic Varicosities in Mouse MS
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From each brain, four consecutive VAChT-stained slices containing the MS were sampled. Using the rat brain atlas of Paxinos and Watson [25] , the sections were level-matched across all animals included in the analysis. The sections were visualized using an Axio Scope.A1 microscope equipped with a Leica EC3 color digital camera. In each section, two photomicrographs were taken with a 100× objective lens. Using the Fiji image-processing software (http://rsb.info.nih.gov/ij/ ), two non-overlapping counting frames of 1,274 µm2 each were applied to each image, and the number and cross-sectional area of all varicosities that fell inside each frame were calculated. The varicosity densities obtained were standardized to an arbitrary area of 30,000 µm2 and averaged over all sections per animal. Cross-sectional areas ranging from .11 to .21 µm2 were included.
4
Immunohistochemical Staining of Immune Cells
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For immunohistochemical stainings, 2 μm tissue sections were incubated in EnVision Flex Target Retrival Solution (Low or High pH; Dako, Santa Clara, CA), followed by incubation with primary antibodies recognizing CD68 (1:200; Abcam, Cambridge, UK), CD3 (1:2000; Santa Cruz Biotechnology, Heidelberg, Germany), GR1 (1:200; BD Biosciences, Heidelberg, Germany), or the GR (1:200; Santa Cruz Biotechnology) for 30 minutes at room temperature. Polymeric secondary antibodies coupled to HRP (ImmPRESS HRP Polymer Detection Kit; Vector Laboratories, Burlingame, CA) and DAB (Dako) were employed to visualize sites of immunoreactivity (dark brown staining). Hematoxylin was used for counterstaining and photomicrographs were acquired with a Leica Axio Scope A1 microscope.
5
Birefringence-Based Fiber Orientation Analysis
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Birefringence is an optical property
of fibers which was determined from three selected filaments with
a Zeiss Axio Scope A1 microscope and a Leica B 5λ-Berek tilting
compensator. Titer was measured three times with the vibroscope, permitting
1.5% variation to exclude artifacts, and the diameter was calculated
assuming a density of 1.5 g/mL.37 During
the measurement, the filaments are tensioned between two pieces of
double-sided tape on a microscope slide. The optical retardation is
determined in triplicate from a selected spot. Birefringence is defined
as the retardation divided by the diameter; according to the literature,
a birefringence of 0.062 was defined as equivalent to 100% orientation.38 (link),39 (link)
of fibers which was determined from three selected filaments with
a Zeiss Axio Scope A1 microscope and a Leica B 5λ-Berek tilting
compensator. Titer was measured three times with the vibroscope, permitting
1.5% variation to exclude artifacts, and the diameter was calculated
assuming a density of 1.5 g/mL.37 During
the measurement, the filaments are tensioned between two pieces of
double-sided tape on a microscope slide. The optical retardation is
determined in triplicate from a selected spot. Birefringence is defined
as the retardation divided by the diameter; according to the literature,
a birefringence of 0.062 was defined as equivalent to 100% orientation.38 (link),39 (link)
6
Histological Evaluation of Intestinal Tissue
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Tissue biopsies were fixed for 48 hours in 4% PFA (Carl Roth, Karlsruhe, Germany) at room temperature and subsequently dehydrated and embedded in paraffin. 2 µm sections were prepared and stained with hematoxylin and eosin (H&E) or by Periodic acid-Schiff reaction (PAS) according to standard protocols. Photomicrographs were acquired using a Leica Axio Scope A1 microscope (Wetzlar, Germany). Histopathological scores were determined in a blinded manner by evaluating four criteria in ten fields per section [42 (link)]: (A) villous blunting/flattening (0 = none, 1 = yes; 20× magnification), (B) number of apoptotic cells (40× magnification), (C) grade of inflammation (0 = none, 1 = mild, 2 = moderate without abscess, 3 = presence of abscess, erosions or ulcer; 20× magnification), (D) edema (0 = none, 1 = yes; 20× magnification). The total histopathological score assigned to each section was calculated as the sum of individual scores obtained for each criterion in all fields. The average number of goblet cells per villus was determined by counting PAS-positive cells in a total of ten villi at 40× magnification.
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