For mucin and goblet cell quantification, 8 µm sections of tissue were deparaffinized three times for 5 min using HistoChoice® Clearing Agent (VWR, catalog #97060-932) and rehydrated. Alcian Blue Periodic Acid Schiff (AB/PAS) staining was performed following manufacturer protocol (Fisher Scientific, Waltham, MA, USA, #88043, #88016). Alcian blue stains highly acidic mucopolysaccharides blue and PAS stain neutral-acidic mucopolysaccharides pink. Intestinal goblet cells contain both neutral and acidic mucins which can be determined by a deep purple stain within the intestinal villi structure. Images were blindly scored on a scale from 1–5 for neutral-acidic mucins (pink), highly acidic mucins (blue), and a combination of mucins (deep purple) along the intestinal villi. Scoring was quantified on a 5-point scale based on the intensity of pink, blue, and purple, with 5 being the most intense, and combined for a total of a 15-point scale. Goblet cells were quantified per 100 µm of intestinal villi. A minimum of 4 sections per slide with a minimum of 4–5 locations per section were imaged, and n = 3–5 per group were used for analysis. Histological endpoints were imaged at 20x and 40x magnification with a bright field microscope and luminal contents were excluded in the analysis.
Histochoice clearing agent
Manufactured by Avantor
Sourced in United States
HistoChoice Clearing Agent is a laboratory reagent used in the preparation of tissue samples for microscopic examination. It is a clearing agent that enhances the transparency of fixed and processed tissue, allowing for better visualization of cellular structures and tissue architecture during histological analysis.
Automatically generated - may contain errors
Lab products found in correlation
Hematoxylin qs, by Vector Laboratories (2 mentions)
Vectamount, by Vector Laboratories (2 mentions)
Mab6158, by R&D Systems (1 mentions)
Antigen unmasking solution, by Vector Laboratories (1 mentions)
Ab8887, by Abcam (1 mentions)
Histochoice mb tissue fixative, by Avantor (1 mentions)
Ix70 inverted microscope, by Olympus (1 mentions)
Dpx mounting medium, by Merck Group (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
6 protocols using histochoice clearing agent
1
Quantifying Intestinal Mucins and Goblet Cells
2
Small Intestine Tissue Preparation
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Mice were anesthetized with Euthasol and euthanized by exsanguination via cardiac puncture within 24 h of final exposure. The small intestine was immediately excised and weighed (data not shown). Luminal contents were flushed, collected, snap-frozen, and stored in −80 °C. The small intestine was dissected into three portions: duodenum, jejunum, and ileum. A 2 cm section of each the proximal portion of the duodenum, the proximal portion of the jejunum, and the distal portion of the ileum were collected and immediately fixed in zinc formalin buffer (Sigma–Aldrich, St. Louis, MO, USA, catalog #Z2902). The remaining portions of the small intestine were snap frozen and stored at −80 °C for later analysis. Fixed intestinal portions were dehydrated in ethanol and treated in HistoChoice® Clearing Agent (VWR, Wayne, PA, USA, catalog #97060-932). Tissues were then embedded in paraffin blocks and sectioned at 8 µm.
3
Immunohistochemistry of Adrenal and Brown Fat
4
Fixation and Imaging of Embryos
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Approximately 105 fresh embryos were fixed in Histochoice Tissue Fixative MB (Amresco, Solon, OH) for 2 h and cleared in Histochoice Clearing Agent (Amresco, Solon, OH). 4',6-Diamidino-2-phenylindole (DAPI) was added to a final concentration of 0.1 μg/ml. The stained eggs were transferred to a slide, covered with a coverslip and sealed with clear nail polish. Eggs were viewed with an Olympus IX70 inverted microscope using a 40× (NA 0.75) objective lens and settings for both Nomarski (Differential Interference Contrast) and fluorescent DAPI imaging. Squash preparations to count nuclei of developing embryos were prepared as reported previously [16 (link)].
5
Histological Analysis of Intestinal Tissues
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Sections of the colon and ileum were de-waxed using three 10 min incubations in Histochoice clearing agent (Amresco, H103). Sections were then rehydrated through a series of ethanol concentrations (100, 95, 90, 70%) for 2 min each, then rinsed with water for 5 min. Sections were then placed into Mayer’s Hematoxylin (VWR, 10047005) for 5 min, followed by a 5 min water rinse. Sections were then placed in Eosin (VWR, 10047003) for 30 s, rinsed with water, and dehydrated through a series of ethanol concentrations (70, 90, 95, 100%) for 2 min each. Sections were then placed in two 2 min incubations of Histochoice, then coverslipped with DPX mounting medium (Sigma, 06522). Sections were imaged at 40X using a brightfield microscope (Olympus DP71), and 5 crypt and 5 villi length were measured for each animal (n = 8 for healthy animals and n = 12 for Citrobacter infected animals) using ImageJ software (NIH), and the values were averaged per animal for each tissue type. All measurements were conducted in blind to groups.
6
Immunohistochemistry of FMO3 in Rat Liver
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