A tracer labeled as 70 kDa Rhodamine (RB)-Dextran (RuixiBiotechCo.Ltd, R-TRD-005) was dissolved in artificial cerebrospinal fluid to attain a concentration of 2%. This solution was then injected into the brain parenchymal lesion with a volume of 2 µL, utilizing a stereotactic apparatus, for a duration of 10 min. The injection coordinates were set as follows: anterior/posterior (AP) 1.5 mm; medial/lateral (ML) 1.5 mm; dorsal/ventral (DV) 2.5 mm from the CSF. To prevent any potential backflow, the syringe remained in place for 5 min following each injection. The mice were administered deep peritoneal anesthesia, succeeded by infusion of 0.9% saline via cardiac means, and subsequently perfused with a solution of 4% paraformaldehyde using a perfusion pump (Cole-Parmer, USA). After dissection, the brain was securely immobilized at a temperature of 4 °C for the duration of one night. Subsequently, the brain was sectioned into slices measuring 100 µm in thickness. These slices were utilized to observe the outflow of the tracer through employment of a laser scanning confocal microscope (NIS-elements, AX, Tokyo, Japan).
Perfusion pump
Manufactured by Cole-Parmer
Sourced in United States
The Perfusion Pump is a laboratory device designed to precisely control the flow of fluids or liquids. It is used to deliver a continuous, regulated flow of materials for various applications, such as cell culture, drug delivery, and other research purposes. The pump operates by using a motor to drive a mechanism that precisely controls the flow rate, ensuring accurate and consistent fluid delivery.
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5 protocols using perfusion pump
1
Tracing Cerebrospinal Fluid Dynamics in Brain Lesions
2
Perfusion-based tissue preparation protocol
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Mice were intraperitoneally deeply anesthetized, then transcardially perfused with 0.9% saline by perfusion pump (Cole-Parmer, USA), followed by 4% paraformaldehyde. Forebrains, meninges, and dcNLs were dissected out and post-fixed overnight at 4℃. The meninges were directly unfolded on a glass slide covered with poly-l-lysine for immunofluorescence. For H&E and immunochemical staining, a subset of forebrains was prepared as paraffin sections. Coronal sections at 5 µm were serially cut using a paraffin slicing machine (Leica RM2135, Nussloch, Germany). For analysis of immunofluorescence and TR-d3 diffusion, forebrains and dcLNs were gradient-dehydrated by 20% and 30% sucrose for 24 hours. Tissues were embedded with OTC gel. Then serial sections at 40 µm were cut on a cryostat (Leica CM1900, Nussloch, Germany).
3
Tissue Harvesting and Preservation Protocol
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Following anesthesia, mice were transcardially perfused with 0.9% saline, followed by 4% paraformaldehyde (PFA) by perfusion pump (Cole-parmer, USA). Brain tissues and dcLNs were then post-fixed in 4% PFA overnight and dehydrated in 20% sucrose solution dissolved in 0.01 M PBS for 3 days. Sections were cut at 30 μm on a cryostat (Leica, Wetzlar, Germany) and mounted onto gelatin-coated slides. For CSF tracer experiments, PFA post-fixed forebrain tissues were coronally sliced on a vibratome (Leica) at 100 μm and mounted onto gelatin-coated slides in sequence. For biochemical analyses, anesthetized mice were euthanized by decapitation. The cerebral cortex was promptly dissected. Tissues were flash frozen in liquid nitrogen and stored at − 80 °C awaiting analysis.
4
Histological Examination of Mouse Brains
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Subsequent to deep anesthesia, mice were transcardially perfused with 0.9% saline by perfusion pump (Cole-parmer) for 5 minutes, followed by 4% paraformaldehyde for 12 minutes. Brains were dissected in the mid-sagittal plane, postfixed overnight at 4°C, then dehydrated in a series of graded ethanol solutions and embedded in paraffin. Both half-brains were serially cut into 5-µm sagittal sections using a paraffin slicing machine (Leica RM2135, Nussloch, Germany). Sections from one half-brain were divided into 5 sets for HE staining and Thioflavin-S staining, while serial sections from the other half-brain were used for immunohistochemical stains.
5
Brain Tissue Preparation and Preservation
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After anesthesia, mice were transcardially perfused with 0.9 % saline, followed by 4 % paraformaldehyde by perfusion pump (Cole-parmer, USA). Brains were dissected mid-sagittallly, postfixed overnight at 4 °C, dehydrated in a series of graded ethanol solutions then embedded in paraffin. Sagittal brain sections were serially cut at 5 μm using a paraffin slicing machine (Leica RM2135, Nussloch, Germany), and collected form Lateral −0.96 to −1.92 mm in the mouse brain atlas as landmarks [62 ]. Every tenth section was collected as 1 set, and 10 sets per brain hemisphere were obtained. For biochemical analyses, mice were euthanized by decapitation. The cerebral cortex and hippocampus were promptly dissected. Tissues were flash frozen in liquid nitrogen and stored at −80 °C until analysis.
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