Mice were anesthetized with ketamine-xylazine injectables and perfused with PBS and then with 4% (w/v) paraformaldehyde in PBS, followed by dissection of the brain for immunofluorescence microscopic examination (23 (link), 59 (link)). Briefly, samples were incubated in PBS containing 0.05% Tween 20 (PBST) and 10% sucrose for 3 hours and then 30% sucrose overnight at 4°C. Brain tissue was then embedded in OCT (Tissue Tech) at –80°C and processed for conventional cryosectioning. Frozen sections (30-μm-thick) were treated with cold ethanol (–20 °C), followed by 2 rinses in PBS, blocking with 3% BSA in PBST, and double labeling with 2 antibodies (Supplemental Table 3 ). After 3 washes in PBST, the sections were further incubated with Cy2 and Cy5 (Jackson ImmunoResearch Laboratories). The samples were mounted and observed under an Olympus IX81 fluorescence microscope. Counting analysis was performed using Olympus Microsuite V software with the help of a touch counting module.
Cy2 and cy5
Manufactured by Jackson ImmunoResearch
Cy2 and Cy5 are fluorescent dyes commonly used in biological research. Cy2 has an absorption maximum at 489 nm and an emission maximum at 506 nm, while Cy5 has an absorption maximum at 649 nm and an emission maximum at 666 nm. These dyes can be used to label proteins, nucleic acids, or other biomolecules for detection and analysis.
Automatically generated - may contain errors
4 protocols using cy2 and cy5
1
Immunofluorescence Microscopic Analysis of Mouse Brain
2
Immunofluorescence Analysis of Mouse Brain Tissue
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Example 17
Mice were anesthetized with ketamine-xylazine injectables and perfused with PBS and then with 4% (w/v) paraformaldehyde in PBS followed by dissection of the brain from each mouse for immunofluorescence microscopy (23, 59). Briefly, samples were incubated in PBS containing 0.05% Tween 20 (PBST) and 10% sucrose for 3 h and then 30% sucrose overnight at 4° C. Brain was then embedded in O.C.T (Tissue Tech) at −80° C., and processed for conventional cryosectioning. Frozen sections (30 μm) were treated with cold ethanol (−20° C.) followed by two rinses in PBS, blocking with 3% BSA in PBST and double-labeling with two antibodies (table S3). After three washes in PBST, sections were further incubated with Cy2 and Cy5 (Jackson ImmunoResearch Laboratories, Inc.). The samples were mounted and observed under an Olympus IX81 fluorescence microscope. Counting analysis was performed using Olympus Microsuite V software with the help of touch counting module.
3
Immunofluorescence Microscopy of Mouse Brain
4
Brain Immunofluorescence Microscopy Protocol
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Mice were anesthetized with ketamine–xylazine mix solutions and perfused with PBS and then with 4% paraformaldehyde (w/v) in PBS, followed by dissection of the brain for immunofluorescence microscopic examination [50 (link),51 (link),52 (link)]. Briefly, the dissected brains were incubated in 10% sucrose for 3 h, followed by 30% sucrose overnight at 4 °C. Then, the brains were embedded in optimal cutting temperature medium (Tissue Tech) at −80 °C and processed for conventional cryosectioning. Frozen sections (40 µm thickness) were treated with cold ethanol (−20 °C), washed with PBS, blocked with 2% BSA in PBST and double labeled with two primary antibodies (Table 1 ). After three washes with PBST, sections were incubated with Cy2 and Cy5 (Jackson ImmunoResearch Laboratories). The sections were mounted and observed under an Olympus IX81 fluorescence microscope. Counting analysis was performed using Olympus Microsuite V software with the help of a touch counting module.
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