The samples were fixed in 10 % neutral buffered formalin, decalcified in Shandon TBD-2 Decalcifier (TBD; Thermo Scientific, Kalamazoo, MI, USA) and embedded in paraffin. Cartilage sections (5 μm thick) were stained with safranin O-fast green using an established protocol [50 (link)] to detect the glycosaminoglycan (GAG) positive matrix that is typical for the hyaline cartilage and normally stains red. Light microscopy images were taken at 4× magnification with Olympus BX63 microscope (Olympus, Japan) equipped with Olympus DP72 CCD camera using CellSens Dimension imaging software (Olympus, Japan). The images were analysed using Image J software. Previously described image processing protocol was adapted to quantify the amount of GAG distribution in each section [51 (link)]. Briefly, red colour intensity (RCI) was calculated in the entire cross-sectional slice area by obtaining red, green, and blue (RGB) image planes in a scale of 256 values (black = 0). The fraction of red (RF) was defined as the ratio of the R component to the sum of the R, G, and B components: RF = R/(R + G + B) and expressed as a percentage. Sections were additionally evaluated by 3 blinded observers using histological-histochemical grading system proposed by Mankin et al. [52 (link)]. Fresh articular cartilage received the score of 0, whereas higher score indicated more deteriorated cartilage.
Shandon tbd 2 decalcifier
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Shandon TBD-2 decalcifier is a laboratory equipment designed for the removal of calcium deposits from tissue samples prior to histological processing. It functions by chemically decalcifying the specimens to facilitate sectioning and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Dp72 ccd camera, by Olympus (1 mentions)
Bx63 microscope, by Olympus (1 mentions)
Cellsens dimension imaging software, by Olympus (1 mentions)
Z fix, by Anatech (1 mentions)
Dm 1000 led microscope, by Leica (1 mentions)
Rm2255 rotary microtome, by Leica (1 mentions)
Application suite software, by Leica (1 mentions)
6 protocols using shandon tbd 2 decalcifier
1
Quantifying Cartilage GAG Content
2
Evaluating Graft Resorption Rates
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The samples were decalcified using a Shandon TBD-2 DECALCIFIER (Thermo Scientific, USA) and embedded in paraffin. The five tissue sections (100 m away from each section) obtained in 4-µm thickness were stained with hematoxylin and eosin. The samples were thoroughly observed under a microscope, and the regions involving proximal and distal host bone in the slides were photographed. Residual graft areas (mm2) were then calculated using a digital image analyzer (Image Partner Software; Saram soft, Korea) to evaluate the rate of resorption of the grafts.
3
Quantitative Histological Analysis of Intervertebral Disc Degeneration
4
Decalcification and Histological Analysis of Mouse Heads
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After nasal washes, mouse heads were placed into formalin for 24 h before being placed in a Shandon TBD-2 decalcifier (Thermo Scientific, Kalamazoo, MI) for 4 days. Decalcified heads were placed in formalin for 2 days, followed by twice-daily washes with PBS for an additional 4 days. Samples were washed with and placed in 70% ethanol prior to standard histological processing and embedded in paraffin wax. Cross-sectional slices (5 μm) were mounted on slides and stained with hematoxylin and eosin according to standard protocols. Slides were randomized and scored in a blind fashion. The integrity of the epithelial lining was measured by quantifying the area of intact/disrupted epithelium using ImageJ.
5
Immunofluorescence and Histological Analysis of Tissue Samples
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Mice were perfused with saline and 4% paraformaldehyde, followed by fixation in paraformaldehyde. The tissue was cut into free floating sections (brain parenchyma) or 10 μm frozen sections on slides (dural metastases). The same antibody clones were used as for flow cytometry. Images were acquired with AxioImager Z1 fluorescence microscope equipped with AxioCam MRc5 digital camera using AxioVision Rel. 4.7 software (Zeiss). Fixed whole heads were decalcified in Shandon TBD-2 decalcifier (Thermo Scientific) for 24 hours. Coronal sections of whole heads (10 um) were cut onto slides and stained with H&E or Verhoeff's Elastic Stain Kit (American MasterTech) according to the manufacturer's protocol.
6
Histological Analysis of Mandibular Symphysis in ENT1-/- Mice
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Following imaging, the heads of 12 age-matched wild-type and ENT1−/− mice at 3 and 6 months-of-age (n = 3 mice per genotype per age) were decalcified with Shandon™ TBD-2 decalcifier (Thermo Fisher Scientific, Waltham, MA) for 4 days. Heads were then embedded in paraffin for serial sectioning (5 μm thick slices) in the transverse anatomical plane RM2255 Rotary Microtome (Leica Biosystems Nußloch GmbH, Nußloch, DEU). Sections of the mandibular symphysis from wild-type and ENT1−/− mice were stained by haematoxylin and eosin (H&E) or Lillie's modified trichrome. Light micrographs were acquired using a Leica DM1000 LED microscope, Leica DFC295 digital colour camera, and Leica Application Suite software (Version 3.8.0, Leica Microsystems GmbH, Wetzlar, DEU). One of the authors (PKK) with specialized training in veterinary anatomic pathology, reviewed the stained sections and documented histological features of the mandibular symphysis in wild-type and ENT1−/− mice.
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