To measure the extent of axonal regeneration, NH was performed. The median nerve was harvested at time of sacrifice, fixed in 2% glutaraldehyde, post-fixed with 1% osmium tetroxide, and embedded in Araldite® 502 (Polyscience). A 10-mm segment of median nerve, 5 mm distal to the repair site (at the level of the rodent wrist) was cross-sectioned and stained with 1% toluidine blue for light microscopy. Digital images of the median nerve were then taken using an unbiased sampling method of non-overlapping regions of the entire cross-section. Total number, density, diameter, and g-ratio of myelinated axons was quantified using Image J software. For each sample, a minimum of 200 myelinated axons was measured and the average was counted as n = 1.
Araldite 502
Manufactured by Polysciences
Sourced in Sweden, United States
Araldite 502 is a two-part epoxy resin system. It is a clear, low viscosity, room temperature curing adhesive. Araldite 502 provides excellent adhesion to a variety of substrates and exhibits good mechanical properties after curing.
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9 protocols using araldite 502
1
Quantifying Axonal Regeneration in Nerve Repair
2
Quantitative Nerve Histomorphometry
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The axial 1 cm of the nerve distal to the injured site was isolated and fixed at 4°C with 3% glutaraldehyde (Polysciences Inc., Warrington, PA), washed in 0.1 M phosphate buffer (pH 7.2), post-fixed with 1% osmium tetroxide (Fisher Scientific, Pittsburgh, PA), dehydrated in graded ethanol solutions, and embedded in Araldite 502 (Polysciences Inc.). Axial semi-thin sections, with 1-μm-thick nerve specimens, taken at a 5-mm distance from the injured site, were stained with 1% toluidine blue for histomorphometric analysis. Binary image analysis was used for semi-automated quantitative analysis of multiple components of nerve histomorphometry; this was performed by an observer blinded to experimental conditions [33 (link), 63 (link)]. Total myelinated fiber counts were measured based on 6 representative fields at 1000 × magnification. Fiber width, axon width, total fiber area, fiber area, myelin area, axon area, and fiber debris area was calculated and compared.
3
Histological Analysis of Immunized Eye Tissues
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Eyes were harvested at early (40–90 days), intermediate (100–200 days) and late (over 200 days) recovery times post-immunization (p.i.). Right eyes were used for histology and were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1M PO4 buffer (pH = 7.4) overnight and dehydrated in graded ethanol and propylene oxide. After polymerization in a resin mixture containing Polybed 812 (Polysciences) and Araldite 502 (Polysciences), semi-thin (0.7 µm) sagittal sections of each eye were stained with toluidine blue and analyzed for histopathology with light microscopy using a Zeiss microscope (equipped with an AxioCam digital camera) using a 63x oil-immersion lens.
4
Evaluating Nerve Regeneration Post-Injury
5
Peripheral Nerve Histomorphometric Analysis
6
Quantitative Nerve Histomorphometry Analysis
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In brief, the axial 1 cm of the nerve distal to the injured site, as well as of the contralateral naïve nerve, for each subgroup (crush nerve + ADSC-exo, crush nerve + ADSC-F-exo, and nerve crush control, n = 6 for each subgroup) was isolated and fixed at 4 °C with 3% glutaraldehyde (Polysciences Inc., Warrington, PA, USA), washed in 0.1 M phosphate buffer (pH 7.2), post-fixed with 1% osmium tetroxide (Fisher Scientific, Pittsburgh, PA, USA), dehydrated in graded ethanol solutions, and embedded in Araldite 502 (Polysciences Inc.). Axial semi-thin sections (1-μm-thick nerve specimens), obtained at a 5-mm distance from the injured site, were stained with 1% toluidine blue for histomorphometric analysis. Binary image analysis was performed for semi-automated quantitative analysis of multiple components of nerve histomorphometry in a blinded manner [71 (link)]. Total myelinated fiber counts were measured based on six randomly selected fields at 1000 × magnification. The fiber count, fiber width, axon width, fiber area, axon area, myelin area, and total fiber area were calculated.
7
Mouse Eye Histology: Optic Nerve Analysis
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After treatment, intact mouse eyes were enucleated, fixed in Karnovsky’s fixative (2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer) for 60 min, removed the cornea and further fixed in Karnovsky’s fixative overnight. Then the eyes were fixed in 1% osmium tetroxide, dehydrated in graded ethanol and then propylene oxide. Next, eyes were transferred to a plastic resin mixture containing Polybed 812 and Araldite 502 (Polysciences) with polymerizer. After polymerization, five to ten sections of 1 μm-thickness were sectioned sagittally, passing through the optic nerve head. Slides were placed 1–2 drops of 1% toluidine blue in 1% sodium borate on a hot plate for 20 s and then rinsed with a gentle stream of distilled water to wash off the excess stain. Slides were covered by coverslips and visualized under the microscope. Pictures were taken on either side of the optic nerve head using a microscope, and the thickness of the ONL and INL was measured at 200 μm from the edge of the optic disc using ImageJ 1.48v software (National Institutes of Health, MD).
8
Femoral Nerve Anatomy and Branching
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Four fresh-frozen cadaveric lower limbs were dissected for anatomical analysis of the femoral nerve and its branches to the sartorius. Surface anatomy landmarks, branching patterns, and nerve fiber counts of terminal branches greater than 1 mm in diameter were recorded. The methodology of nerve sample processing and histological analysis used in this study has been previously reported. 16, 17 Briefly, the nerves were fixed in 3% glutaraldehyde solution in 0.1 M phosphate-buffered saline (pH 7.2) at 4°C prior to sectioning. The tissues were then postfixed with 1% osmium tetroxide and serially dehydrated in ethanol. Specimens were embedded in Araldite 502 (Polysciences Inc.), sectioned into 1-µm-thick cross-sections, and stained with 1% toluidine blue and evaluated via light microscopy. Microscopic images were analyzed with an automated digital image analysis system linked to histomorphometry software to quantify the total nerve fiber count.
9
Histomorphometric Analysis of Myelinated Nerve Fibers
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Harvested nerve segments were fixed at 4°C with 3% glutaraldehyde (Polysciences Inc.), washed in 0.1-M phosphate buffer, postfixed with 1% osmium tetroxide (Fisher Scientific), dehydrated in graded ethanol solutions, and embedded in Araldite 502 (Polysciences Inc.). Semi-thin sections (1 μm thick) were stained with 1% toluidine blue for histomorphometric analysis. Sections were examined using an imaging microscope (Carl Zeiss) and analyzed with ImageJ by experimenters (M.Y. and L.L.) blind to the source nerve to determine the numbers of myelinated fibers and area of total myelinated fibers.
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