Following an overdose injection of sodium pentobarbital, each animal was transcardially perfused with approximately 50 ml of 0.9% NaCl followed by 50 ml of 4% paraformaldehyde solution (PFA; pH 9.5). The brains were post-fixed in 4% PFA for 24–48 hours at 4°C after which they were embedded in 3% Type I-B agarose (Sigma-Aldrich) prior to sectioning. Four series of coronal sections were sliced at 50-μm thickness with a Compresstome (VF-700, Precisionary Instruments, Greenville, NC) and prepared for processing.
Type 1 b agarose
Manufactured by Merck Group
Sourced in United States
Type I-B agarose is a laboratory gel material used for various separation and purification techniques. It is derived from red seaweed and serves as a medium for electrophoresis, chromatography, and other analytical procedures. The core function of Type I-B agarose is to provide a stable, porous matrix for the movement and separation of macromolecules, such as proteins and nucleic acids, based on their size and charge characteristics.
Automatically generated - may contain errors
Lab products found in correlation
Neurotrace 435 455, by Thermo Fisher Scientific (3 mentions)
Stereotaxic apparatus, by GE Healthcare (1 mentions)
Vaporizer, by GE Healthcare (1 mentions)
Nanoject 3, by Drummond (1 mentions)
As 2300, by Vector Laboratories (1 mentions)
Naio4, by Merck Group (1 mentions)
Lidocaine, by Merck Group (1 mentions)
Risperidone, by Thermo Fisher Scientific (1 mentions)
7 protocols using type 1 b agarose
1
Perfusion, Fixation, and Sectioning of Brain Tissue
2
Perfusion, Fixation, and Sectioning of Brain Tissue
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Following an overdose injection of sodium pentobarbital, each animal was transcardially perfused with approximately 50 ml of 0.9% NaCl followed by 50 ml of 4% paraformaldehyde solution (PFA; pH 9.5). The brains were post-fixed in 4% PFA for 24–48 hours at 4°C after which they were embedded in 3% Type I-B agarose (Sigma-Aldrich) prior to sectioning. Four series of coronal sections were sliced at 50-μm thickness with a Compresstome (VF-700, Precisionary Instruments, Greenville, NC) and prepared for processing.
3
Perfusion-fixed Brain Sectioning and Tracing
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The perfusion‐fixed brains were post‐fixed in 4% PFA for 24–48 h at 4°C, after which they were embedded in 3% Type I‐B agarose (Sigma‐Aldrich) prior to serial sectioning. Four series of coronal sections were sliced at 50‐μm thickness with a Compresstome (VF‐700, Precisionary Instruments, Greenville, NC), and stored in cryoprotectant at –20°C until further processing for detection of axonal tracers and Nissl staining. Immunocytochemical detection of PHAL in free‐floating sections was accomplished with a polyclonal rabbit anti‐PHAL primary antibody (diluted 1:5,000; Vector Labs #AS‐2300), followed by a polyclonal donkey anti‐rabbit secondary antibody conjugated to AlexaFluor 488 (diluted 1:1,000; Jackson ImmunoResearch #711‐545‐152). Detection of FG, CTB‐647, AAV‐tdTomato, and AAV‐GFP tracers was accomplished without immunocytochemistry. Sections were counterstained with a fluorescent Nissl stain (NeuroTrace 435/455, diluted 1:500; Invitrogen, #N21479) and then were mounted and coverslipped using 65% glycerol mountant.
4
Retrograde Tracing of Mouse Brain Circuits
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On the day of the experiment, mice were deeply anesthetized and mounted into a Kopf stereotaxic apparatus where they were maintained under isofluorane gas anesthesia (Datex-Ohmeda vaporizer). For quadruple retrograde tracing experiments, 50nl of retrograde tracers were individually pressure-injected via glass micropipettes at a rate of 10nl/min (Drummond Nanoject III). All injections were placed in the right hemisphere.
After 4-6 days post-surgery, each mouse was deeply anesthetized with an overdose of Euthasol (pentobarbital) and trans-cardially perfused with 50ml of 0.9% saline solution followed by 50ml of 4% paraformaldehyde (PFA, pH 9.5). Following extraction, brain tissue was post-fixed in 4% PFA for 24-48hr at 4°C. Fixed brains were embedded in 3% Type I-B agarose (Sigma-Aldrich) and sliced into four series of 50μm thick coronal sections using a Compresstome (VF-700, Precisionary Instruments, Greenville, NC) and stored in cryopreservant at −20°C. All sections were stained with Neurotrace 435/455 (Thermo Fisher Cat# N21479) for 2-3 hours to visualize cytoarchitecture. After that, sections were mounted onto glass slides and cover slipped using 65% glycerol.
After 4-6 days post-surgery, each mouse was deeply anesthetized with an overdose of Euthasol (pentobarbital) and trans-cardially perfused with 50ml of 0.9% saline solution followed by 50ml of 4% paraformaldehyde (PFA, pH 9.5). Following extraction, brain tissue was post-fixed in 4% PFA for 24-48hr at 4°C. Fixed brains were embedded in 3% Type I-B agarose (Sigma-Aldrich) and sliced into four series of 50μm thick coronal sections using a Compresstome (VF-700, Precisionary Instruments, Greenville, NC) and stored in cryopreservant at −20°C. All sections were stained with Neurotrace 435/455 (Thermo Fisher Cat# N21479) for 2-3 hours to visualize cytoarchitecture. After that, sections were mounted onto glass slides and cover slipped using 65% glycerol.
5
Perfusion-Fixed Brain Sectioning and Labeling
6
Agarose-Embedding for Organ Imaging
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To prepare the embedding medium, oxidized agarose was prepared by adding agarose Type I-B (Sigma-Aldrich, USA) to a 10 mM NaIO4 (Sigma-Aldrich, USA) solution and stirred for 2–3 h at room temperature without exposure to sunlight. Then, the solution was repeatedly washed and resuspended in PBS to increase the final concentration to 2%–6%. To form the final sample, the organs were pat-dried and placed in a cubic silicone mold filled with melted oxidized agarose. The mold was then placed in a 55–65°C water bath for 0.5 h until the surfaces of the organs were fully crosslinked with agarose. Then, the mold and organs were left at room temperature for 0.5 h to allow the agarose to solidify. Finally, the agarose-embedded organs were separated from the mold and stored in PBS at 4°C before imaging.
7
Polymeric Microparticles for Drug Delivery
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The PLGA was obtained from Evonik (Darmstadt, Germany) under the commercial name Resomer® RG. PLGA grades of 50:50 lactic to glycolic acid ratio and of different molecular weights (Mw) and either acid- or ester-terminated were obtained: Resomer® RG 503H (acid-terminated, Mw 24 000–38 000); Resomer® RG 504 (ester-terminated, Mw 38 000–54 000); and Resomer® RG 505 (ester-terminated, Mw 54 000–69 000). Ethyl cellulose was obtained under the commercial name EthocelTM Standard 7 Premium from Dow (Midland, MI, USA). Risperidone and flurbiprofen were obtained from Acros Organics (Geel, Belgium). Lidocaine and agarose type I-B were obtained from Sigma-Aldrich. All other chemicals were of analytical grade or higher.
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