1.1.1.

Dissection and fixation of embryonic, postnatal, and adult leptomeninges

Here, we describe methods for dissection of leptomeninges (collective term for combined pia and arachnoid layers) for ages embryonic days (E)14-18 and postnatal days (P)0 through adulthood (Fig. 2). All meningeal fibroblasts are labeled by GFP in the Col1a1-GFP mouse line. To assay for proliferating cells, we use an EdU assay. EdU is a thymidine analog that is incorporated into DNA during replication in cells in S-phase and can be detected using fluorescent probes.²¹ If completing EdU detection steps, administer EdU ($2.5\mathrm{mg}/\mathrm{mL}$) via interperitoneial injection prior to anesthetization (embryonic: $150\mu \mathrm{L}$ into pregnant dam, P0-P21: $50\mu \mathrm{L}$, adult: $100\mu \mathrm{L}$) [Figs. 2(a.i) and 2(a.ii)]. We allow EdU to circulate for 2 h, as this is the amount of time it takes for EdU to fully circulate and clear from a mouse’s body. However, note that the following methods are compatible with other assay configurations, including varied circulation time and different cell cycle probes (i.e., BrdU, Ki67). For embryonic timepoints, dissect embryos using established methods from pregnant Col1a1-GFP dams and anesthetize using cryoanesthesia until unresponsive, then decapitate [Fig. 2(a.i.a)]. For P0-21 and adult mice, euthanize Col1a1-GFP mice using age-appropriate methods [Fig. 2(a.ii.a)]. To remove blood containing immune cells that can be immunoreactive, mice can be transcardially perfused with PBS (2 to 5 mL for P0-10, 10 mL for P11-21, and 20 mL for adults) prior to brain dissection. To dissect embryonic brains, make a single incision laterally beginning at the back of the head and move anteriorly toward the eye [Fig. 2(a.i.b), dotted line]. Lift the skin and calvarium dorsally, peeling the tissue away from the surface of the brain [Fig. 2(a.i.b), arrow]. Note that earlier embryos (<E16) will have less ossification and the dura/calvarium will come off as a mesenchymal layer. For postnatal and adult mice, decapitate and cut away the scalp by a midline excision caudal to rostral, from between the ears to immediately superior to the eyes and pull skin laterally to expose the calvarium [Fig. 2(a.ii.b)]. Carefully remove the calvarium to maintain the integrity of the dura mater (adhered to the underside of the calvarium) for analyses (if desired) using previously published methods for dura whole mount labeling.²² To achieve this, insert fine surgical scissors into the foramen magnum and guide them anteriorly around the cortices toward the olfactory bulb [Fig. 2(a.ii.c)]. Repeat this process on both sides of the skull, with the excision meeting immediately superior to the olfactory bulb. Lift the calvarium away using forceps by pulling upward from the base of the head, exposing the underlying brain [Fig. 2(a.ii.c)]. Using a spatula, gently lift the brain up to remove and place it in a 10-cm plate containing 1× PBS [Figs. 2(a.i.c) and 2(ii.d)]. Complete the isolation of the leptomeninges using a dissection microscope.

Fig. 2

Dissection, fixation, and immunofluorescent staining of embryonic, postnatal, and adult leptomeninges. (a) Graphical description of brain dissections from (i) embryonic and (ii) postnatal and adult mice. (b) Depiction of leptomeninges loosening and removal steps for (i) embryonic and postnatal and (ii) adult leptomeninges. (c) Workflow for immunofluorescent staining and mounting of leptomeninges. Brain dissection images are aged postnatal day (P)10 with the exception of (b.ii.c), which is a 9-month old adult.

To loosen the leptomeninges from the surface of the cerebrum, use fine forceps to gently pull the meninges away from the brain [Fig. 2(b)]. This is done on both cerebral hemispheres while leaving the meninges attached to the brain near the midline—for fetal and postnatal brain dissections it may help to hemisect the brain prior to leptomeninges loosening [Fig. 2(b.b)]. Loosening is done to ensure that the leptomeninges do not adhere too tightly to the surface of the brain during the fixation step, permitting removal of the cerebral leptomeninges as an intact structure following fixation. For embryonic and postnatal brains, begin loosening using forceps starting at the lateral incision spot made during the calvarium removal step [Fig. 2(b.i.c), red dotted line]. Note that this incision will not be made on early embryonic brains (<E16) due to the location of the incision. Move the forceps under the meninges and pull them away from the surface of the brain, moving dorsally toward the midline [Fig. 2(b.i.c), dotted arrows]. Do not remove the meninges completely—keep them attached to the brain near the midline [Fig. 2(b.i.c), arrowheads]. Pull the leptomeninges back over the surface of the cerebrum to prevent wrinkles in the leptomeninges. For adult mice, remove the cerebellum and brain stem and hemisect the two hemispheres. Remove the thalamus, exposing the cerebrum and hippocampus. Starting at the back of the cerebrum [Fig. 2(b.ii.c)] use fine forceps to slowly loosen a piece of the cerebral leptomeninges that is about ¼ to ¾ of the cerebrum surface. To better visualize the cerebrum surface during meninges removal, cut the hemisphere in the transverse plane and loosen meninges pieces from ventral and dorsal cerebrum separately [Fig. 2(b.ii.a)]. Unlike the embryonic and early postnatal brains, the adult leptomeninges will have some hole artifacts made by the loosening process. Of note, this process of leptomeninges removal is especially difficult for brains aged P10 and beyond, possibly due to the leptomeninges becoming thinner with increased brain size, and increasing adherence to the brain. We have found that from ∼P10 to $\sim 4$ months, it is not possible to get large, intact pieces of leptomeninges for whole mount. Instead, it is typical to get small pieces of leptomeninges, usually attached to the middle cerebral artery and primary branches. Once the leptomeninges are loosened, place the brain in 2% paraformaldehyde solution overnight at 4°C then transfer the brain into PBS solution at 4°C (up to 5 days) [Figs. 2(b.i.d) and 2(ii.d)].

Methanol fixation is necessary for certain antibodies, in particular junctional proteins found in pial vasculature and arachnoid barrier layer cells (Table 1). Methanol fix can be substituted for 2% PFA with the following modifications. Following leptomeninges “loosening” of embryonic or postnatal brains [described above, Fig. 2(b)], place individual cerebral hemispheres in methanol for 10 min. Brains will turn white. Carefully remove hemispheres with spatula and place in a large drop of PBS in the lid of a 10 cm plate. Use fine forceps and dissecting scope, position the hemisphere so the leptomeninges can be gently detached and floated into the PBS drop. Once detached [Fig. 2(b.i.e)], the leptomeninges are ready for immunostaining [see below, Fig. 2(c)]. For adult mice, loosen leptomeninges as described above, detach piece and transfer to a glass slide, then float the leptomeninges into a large drop of PBS so the “brain side” faces the glass slide [Fig. 2(b.ii.e)]. Use a dissection scope and fine forceps to untangle (if necessary) and remove PBS using a transfer pipette and twisted Kimwipe and let dry completely (tissue will appear white when dry). Transfer the slide to a plastic container with methanol or use a hydrophobic pen to create a barrier (for fixation on slide); fix for 10 min. Transfer slide to PBS and start immunostaining protocol [see below, Fig. 2(c)].

Table 1

Markers compatible with meningeal whole mount staining.

Complete leptomeninges isolation of embryonic, postnatal, and adult brains after overnight 2% paraformaldehyde (PFA) fixation by placing brains into a 10-cm dish in $1\times$ PBS solution under a dissection microscope. Detach the leptomeninges from both cerebral hemispheres by gently pulling up on the loosened areas until the meninges can be peeled off as one large piece for staining [Fig. 2(b.i.e)]. For adult, transfer the piece of leptomeninges to a glass slide containing a large drop of PBS as described above for methanol fixation [Fig. 2(b.ii.e)]. Once adult leptomeninges are dried on slide, use hydrophobic pen to create box around meninges piece and proceed with immunostaining protocol.

1.1.2.

Immunofluorescent staining protocol

We have optimized a protocol for whole mount immunofluorescent staining and EdU detection on embryonic, postnatal, and adult leptomeninges [Fig. 2(c)]. Antibodies that have been used successfully with these methods are listed in Table 1.

Following dissection of embryonic and postnatal leptomeninges, use fine forceps to transfer each piece into individual wells of a 48-well plate filled with PBS to proceed with staining. For adult leptomeninges, complete staining steps on slides prepared as described above. Recipes for solutions used in this protocol are included in Table 2.

Table 2

Recipes for reagents used in leptomeningeal and choroid plexus whole mount staining.

Block using a solution consisting of 0.1% Triton X-100, 1% bovine serum albumin (BSA), 2% lamb serum, and 0.05% Tween 20 in PBS on a shaker for 1 h at room temperature, adding 100 to $200\mu \mathrm{L}$ of blocking solution per well of a 48-well plate (free-floating embryonic or postnatal leptomeninges) or 100 to $150\mu \mathrm{L}$ per piece (on slide, adult leptomeninges) [Fig. 2(c.i)]. Following this, incubate free-floating leptomeninges on a shaker overnight at 4°C in $100\mu \mathrm{L}$ of primary antibodies (see Table 1) diluted with the primary solution made up of 1% BSA and 0.5% Triton X-100 in PBS [Fig. 2(c.ii)]. Use parafilm to seal the plate and prevent evaporation. For adult tissue on slides, perform this step in a humidified chamber. After overnight incubation in the primary solution, wash the meninges in an excess volume of PBS on a shaker three times for 5 min each wash. Incubate the meninges on a shaker (embryonic and postnatal) or humidified chamber (adult), protected from light, for 1 h at room temperature in $100\mu \mathrm{L}$ of secondary antibodies diluted in secondary solution made up of 1% BSA and 0.05% Triton X-100 [Fig. 2(c.iii)]. Repeat PBS washes on a shaker three times for 5 min per wash. Labeling of cell proliferation via EdU detection is achieved using the Click-iT Plus EdU Alexa Fluor 647 Cell Proliferation Imaging Kit (Thermo Fisher Scientific C10640) [Fig. 2(c.iv)]. Following incubation with EdU detection solution ($100\mu \mathrm{L}$ of solution per well of a 48-well plate for free-floating embryonic or postnatal leptomeninges, or 100 to $150\mu \mathrm{L}$ per adult leptomeninges piece mounted on slide), repeat PBS washes on a shaker three times for 5 min per wash.

After completion of immunostaining and EdU detection, cover-slip adult pieces, or mount embryonic and postnatal leptomeninges on slides following a similar process as described above for adult leptomeninges, transfer leptomeninges to a drop of PBS on a glass slide [Figs. 2(c.v) and 2(vi)] and use fine forceps to remove folds and lay the piece of tissue flat with the “brain side” facing the glass [Fig. 2(c.vii)], then use a transfer pipette and Kimwipe to remove excess PBS [Fig. 2(c.viii)]. Dry tissue in a covered slide box for $\sim 10$ to 15 min [Fig. 2(c.ix)]. Ensure tissue is completely dry (it will appear white) before cover-slipping with a small droplet of mounting media (Fluoromount-G or similar).

Figure 3(a) shows images of meninges from embryonic and postnatal Col1a1-GFP mice whole mounted using these methods, with vessels labeled for PECAM and EdU detection to label to dividing nuclei [Fig. 3(a)]. We are able to clearly distinguish perivascular [Fig. 3(a), inset i, arrowheads] from nonperivascular [Fig. 3(a), inset i, arrows] meningeal fibroblasts and can detect proliferating perivascular cells [Fig. 3(a), inset i, asterisks]. We routinely use these methods with antibody-based staining of meningeal fibroblast markers (full list in Table 1), such as subtype-specific markers RALDH2 to label arachnoid fibroblasts [Fig. 3(b)] and S100a6 to label pial fibroblasts [Fig. 3(c)].⁷ We also use these methods to label junctional proteins expressed by pial vasculature such as Claudin-5 [Fig. 3(e)] and arachnoid barrier cell-enriched junctional proteins E-cadherin [Fig. 3(e)] and Claudin-11 [Fig. 3(f)]. Further, leptomeningeal wholemounts can be used to visualize vasculature and meningeal immune cells like border associated macrophages (Cd206+/Lyve1+) [Fig. 3(g)].

Fig. 3

Immunofluorescent staining and EdU labeling of whole mount leptomeninges. (a) Whole mount leptomeninges from an embryonic day (E)16 Col1a1-GFP mouse. Fibroblasts are labeled by GFP (green), vessels labeled by PECAM (magenta), all nuclei labeled by DAPI (cyan), and proliferating nuclei labeled by EdU (white). Inset (i): arrows indicate nonperivascular fibroblasts labeled by Col1a1-GFP, arrowheads indicate perivascular fibroblasts, asterixis next to arrowhead indicates EdU+ cell. (b), (c) Whole mount leptomeninges from embryonic day (E)17 mice. Vessels are labeled by PECAM (white), and meningeal fibroblasts labeled by markers for (b) arachnoid and perivascular fibroblasts RALDH2 and (c) pial fibroblasts S100a6 (magenta). (d)–(f) Whole mount leptomeninges from postnatal day (P)5-7 mice labeled with junctional markers (d) endothelial Claudin-5 (white), (e) arachnoid barrier cell E-cadherin (magenta), and (f) arachnoid barrier cell Claudin 11 (yellow). (g) Whole mount leptomeninges from embryonic day (E)17 mice, vessels labeled by PECAM (white) and macrophages labeled by Cd206 (magenta) and Lyve-1 (cyan). $\text{Scale bars}=100\mu \mathrm{m}$.