Dfc500 fluorescence microscope

Manufactured by Leica

The DFC500 is a fluorescence microscope designed for high-quality imaging of fluorescent samples. It features a high-resolution digital camera and advanced optics to capture detailed images of fluorescently labeled specimens. The DFC500 is a versatile tool for a wide range of applications in life science research and diagnostics.

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4 protocols using dfc500 fluorescence microscope

Immunofluorescent Labeling of MBP and NF200

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Slices were fixed with 4% PFA, delipidated, and washed three times in PBS, blocked in PBS containing 0.1% Triton X-100 and 5% normal goat serum and incubated with anti-MBP (SMI-99P, Covance, 1:300), and anti-NF200 (N4142, Sigma, 1:250) antibodies overnight at 4 °C. Slices were then washed in PBS and incubated in Alexa Fluor-conjugated secondary antibodies (1:500, Invitrogen) for 2 h. Slices were mounted in Vectashield mounting medium with DAPI (Vector Laboratories) and analyzed using a Leica DFC500 fluorescence microscope.

Luo F., Tran A.P., Xin L., Sanapala C., Lang B.T., Silver J, & Yang Y. (2018). Modulation of proteoglycan receptor PTPσ enhances MMP-2 activity to promote recovery from multiple sclerosis. Nature Communications, 9, 4126.

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Immunohistochemistry of Mouse Brain Sections

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Mice were anesthetized with Avertin and perfused with PBS and 4% paraformaldehyde. Dissected brains were post-fixed in 4% paraformaldehyde overnight at 4 °C and equilibrated in 20% sucrose. 20 μm coronal cryosections were sectioned and pre-treated with Reveal Decloaker Solution (Biocare Medical) for antigen retrieval according to the manufacturer instruction. After blocking, sections were incubated with the primary antibodies and followed by appropriate secondary antibodies conjugated with Alexa florescence 488 or 594. Sections were counter-stained with DAPI (1:1000, Sigma) and mounted with mounting medium (Vector Laboratories). All Images were taken and analyzed using a Leica DFC500 fluorescence microscope.

Luo F., Herrup K., Qi X, & Yang Y. (2017). Inhibition of Drp1 hyper-activation is protective in animal models of experimental multiple sclerosis. Experimental neurology, 292, 21-34.

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Black-Gold II Myelin Staining Protocol

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Black Gold myelin staining was performed according to the manufacturer’s instruction (#AG105, Millipore). Briefly, 20 μm sections were incubated in prewarmed 0.3% Black-Gold II solution at 60 °C for 12 min. After rinsing, sections were incubated with 1% sodium thiosulfate solution for 3 min at 60 °C and rinsed, dehydrated in a series of gradated alcohols, air-dried, and mounted in mounting media. Images were captured from matched areas of stained sections using Leica DFC500 fluorescence microscope. To quantify myelin, the extent of demyelination was scored as previous study (Steelman et al., 2012 (link)): demyelination range from 0% to 10% of the total anatomical structure was scored as 0; 11%–30% represented a score of 1; 31–60% represented a score of 2; 61–90% represented a score of 3; and 91–100% of demyelination represented a score of 4.

Luo F., Herrup K., Qi X, & Yang Y. (2017). Inhibition of Drp1 hyper-activation is protective in animal models of experimental multiple sclerosis. Experimental neurology, 292, 21-34.

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Spinal Cord Immunofluorescence Staining

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Mice were anesthetized with avertin and perfused with PBS and 4% paraformaldehyde (PFA). Spinal cords were dissected and post-fixed in 4% PFA overnight at 4 °C and equilibrated in 20% sucrose. 20 μm-thick sections were pretreated with Reveal Decloaker Solution (RV1000M, Biocare Medical) for antigen retrieval according to the manufacturer’s instructions. After blocking, sections were incubated with primary antibodies overnight at 4 °C and followed by appropriate secondary antibodies conjugated with Alexa fluorescence 488 or 594. The following primary antibodies were used: MBP (SMI-99P, Covance, 1:300), NeuF200 (N4142, Sigma, 1:250), CS56 (C8035, Sigma, 1:250), MMP-2 (AB191677, Sigma, 1:500), laminin (L9393, Sigma, 1:500), CAT301 (MAB5284, Millipore, 1:250), versican (AB1032, Millipore, 1:250), Iba1 (019-19741, WAKO, 1:250), GFAP (MAB360, Millipore, 1:250), Olig2 (AB9610, Millipore, 1:250), CC1 (OP80, Millipore, 1:250), iNOS (#610329, BD Biosciences, 1:250), Arginase-1 (sc-18355, Santa Cruz, 1:50), PDGFRα (Cell signaling, #3174), and NG2 (Millipore, AB5320). For each staining, at least three individual animals/group were examined and images were captured with a Leica DFC500 fluorescence microscope. Staining was quantified using Image J software (US National Institutes of Health, USA). Fluorescence intensity was calculated as percentages of the mean value of the naive controls.

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