Immunofluorescence assay for demyelination, remyelination, and proliferation in an acute cuprizone mouse model

Abstract

Here, we present a protocol to assess demyelination in the corpus callosum of an acute cuprizone mouse model, which is routinely used to induce demyelination for studying myelin regeneration in the rodent brain. We describe the tracing of neural stem cells via intraperitoneal injection of tamoxifen into adult Gli1CreERT2;Ai9 mice and the induction of demyelination with cuprizone diet. We also detail EdU administration, cryosectioning of the mouse brain, EdU labeling, and immunofluorescence staining to examine proliferation and myelination. For complete details on the use and execution of this protocol, please refer to Radecki et al. (2020).¹.

Keywords: Cell Biology; Microscopy; Model Organisms; Molecular/Chemical Probes; Neuroscience; Stem Cells.

Graphical abstract

Figure 1

Perfusion and brain harvest (A) Illustration of the incision point for creating an outlet for the blood and perfusion fluids, and the insertion point for the butterfly needle, demonstrating the position of the needle in the left ventricle. (B) Illustration of mouse brain harvest. Incisions for separating the skin (i-ii), and the bone (iii-vii) to expose the brain. (C) Examples of incompletely perfused (left) vs. completely perfused brains (right).

Figure 2

Cryopreservation and cryosectioning (A) Illustration of the brain embedded in OCT in a tissue mold. (B) Illustration of mounting the frozen tissue block for cryosectioning coronal sections. The chuck is coated with a layer of OCT (i-ii), and the frozen tissue block firmly pressed into the OCT on the chuck (iii). Excess OCT can be carefully trimmed before sectioning (iv). (C) Illustration of olfactory bulb and forebrain sections (from Allen Brain Atlas) on a slide.

Figure 3

Immunolabeling of tissue sections (A) Diagram of a humidity chamber for immunolabeling tissue slides. (B) Tilting the slide for aspirating during washes, as well as using a 200 μL pipet tip on the end of the glass pipet attached to the suction tube to decrease the force of aspiration. (C) Hinging the coverslip onto the slide during mounting.

Figure 4

Immunofluorescence and EdU labeling (A) Immunofluorescent labeling of brain cryosections from mice fed with regular diet (Reg), 0.2% cuprizone diet for 5 weeks (Cup 5wk), and 0.2% cuprizone diet for 5 weeks followed by two weeks regular diet (Cup 5wk + Reg 2wk), showing MBP in the corpus callosum (CC) outlined with white dotted lines. Scale bars = 50 μm, Subventricular zone (SVZ), Lateral ventricle (LV). (B) Examples of immunofluorescent labeling of CC1 expressing mature oligodendrocytes, NG2 expressing oligodendrocyte precursor cells, RFP expressing Gli1 fate mapped cells, and EdU positive proliferating cells in the CC of mice fed with regular diet, cuprizone diet for 5 weeks, and cuprizone diet for 5 weeks followed by two weeks regular diet, with quantification of CC1, NG2, and EdU positive cells in the corpus callosum. N = 3, Data = Mean ± SEM, One-way ANOVA with Tukey’s post-hoc t-test, Scale bars = 25 μm, inset scale bar = 5 μm.