Projections from nucleus basalis of Meynert were traced with Mini-Ruby as described previously (Lai et al. 2012 (link)). Briefly, the engrafted mice were anesthetized with atropine and pentobarbital sodium (intraperitoneal; 0.5 mg/mL and 1 mg/mL, respectively, in saline; 100 µL per gram of body weight). Mini-Ruby (Invitrogen), dissolved at a concentration of 5% in water, was injected into nucleus basalis of Meynert (coordinates: Bregma −0.5 mm, 1.5 mm lateral to midline, depth 3.5 mm) through a sharp electrode. Ten to twelve days after the injection, mice were perfused with 4% paraformaldehyde and their brains were postfixed overnight. Brains were sectioned with a freezing microtome at 40 μm.
Mini ruby
Manufactured by Thermo Fisher Scientific
The Mini-Ruby is a compact and versatile lab equipment designed for various analytical applications. It features a rugged construction and reliable performance to support laboratory workflows.
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6 protocols using mini ruby
1
Mapping Nucleus Basalis Projections
2
Transient Gene Knockdown and Overexpression in Zebrafish
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Transient knock-down of gene expression in zebrafish was performed as described previously
[7 (link)]. We used the following morpholino-antisense oligomers (Gene Tools, Philomath, OR) at a concentration of 0.3 mM each: pax6a MO (5′-TTTGTATCCTCGCTGAAGTTCTTCG-3′) and pax6b MO (5′-CTGAGCCCTTCCGAGCAAAACAGTG-3′)
[53 (link)]. MO oligomers were injected into the yolk cell close to blastomeres at the one-cell or two-cell stage.
For mis-expression experiments full-length pax6 and irx1b was cloned into a pCS2+ vector
[15 (link)] and from this template mRNA was synthesized in vitro (Message Machine Kit, Ambion, Amersham, UK.). Together with rhodamine dextran (MiniRuby, Invitrogen, Carlsbad, CA) as lineage tracer, 350 pg mRNA per embryo was injected into the one-cell stage.
[7 (link)]. We used the following morpholino-antisense oligomers (Gene Tools, Philomath, OR) at a concentration of 0.3 mM each: pax6a MO (5′-TTTGTATCCTCGCTGAAGTTCTTCG-3′) and pax6b MO (5′-CTGAGCCCTTCCGAGCAAAACAGTG-3′)
[53 (link)]. MO oligomers were injected into the yolk cell close to blastomeres at the one-cell or two-cell stage.
For mis-expression experiments full-length pax6 and irx1b was cloned into a pCS2+ vector
[15 (link)] and from this template mRNA was synthesized in vitro (Message Machine Kit, Ambion, Amersham, UK.). Together with rhodamine dextran (MiniRuby, Invitrogen, Carlsbad, CA) as lineage tracer, 350 pg mRNA per embryo was injected into the one-cell stage.
3
Xenopus Microinjection for Gene Manipulation
4
Primary Microglia Isolation and Labeling
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Primary microglia were harvested as previously described [32 (link)]. Briefly, 0-3 day old (d0-3) mice were decapitated and their cerebral cortices were dissected in ice-cold 1x Hank's Buffered Salt Solution (1x HBSS, Thermo Fisher Scientific Inc.). The cortices were trypsinized, homogenized, and plated in DMEM (Thermo Fisher Scientific Inc.) supplemented with 10% FBS, and 1x P/S. Media was replaced on the 3rd day. After 10-15 days, the cultures were treated with lidocaine (Sigma), gently rocked, and microglia were isolated from the supernatant, counted, and resuspended in DMEM supplemented with 1%FBS and 1x P/S. For live imaging of microglia, 20μg/mL Mini-Ruby (Invitrogen) was added to media for 24 hours, after which the media was removed, the culture was rinsed in 1x HBSS, and media was replaced [33 (link)]. All microglia were used for experiments within 48 hours of plating.
5
Zebrafish Plasma and APOC2 Peptide Injection
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Blood was collected by tail amputation from adult male zebrafish at 4 h after the last feeding, and plasma was separated by centrifugation at 2350 g for 10 min. C-II-a and C-II-i peptides (Amar et al., 2015 (link)) were dissolved in PBS (pH 7.4) to a concentration of 2 mg/ml. To ensure visual control of successful injection, zebrafish plasma or APOC2 mimetic peptides were supplemented with 2% (v/v) tetramethylrhodamine-conjugated 10 kDa dextran (0.5 mg/ml miniRuby, Invitrogen, D-3312). For injections, 5 dpf larvae were aligned in 0.5% low-melting-point agarose. A total of 5 nl of plasma or APOC2 mimetic peptides were injected through the cardinal vein above the heart chamber using a FemtoJet microinjector (Eppendorf, Hamburg, Germany). The red signal from fluorescent dextran confirmed successful injection.
6
Tracking Donor Angioblast Migration
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Donor embryos were injected with ctr MO or aplnra/b MO and labeled by the injection of 0.1 ng mini-Ruby (Tetramethylrhodamine, Invitrogen/Life Technologies). At sphere stage, cells were removed from donor embryos and transferred to wild type hosts using a glass capillary. Transplanted angioblasts were identified by transgenic EGFP expression together with mini-Ruby stain. By counting the number of EGFP positive donor angioblasts in the midline vs all EGFP positive donor angioblasts (100%), the percentage of donor angioblasts in the midline was determined for each individual embryo.
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