At the end of the behavioural assessment, 7 weeks for the MPTP model post-lesion. Mice were sacrificed after being anesthetized with isoflurane and perfused with 0.9% saline mixed with 4% paraformaldehyde (PFA; Yakuri Pure Chemicals) in phosphate buffer (PBS) at a pH of 7.4. Organs including the lungs, heart, liver, kidney, ovary, spleen, bladder, spinal cord, and brain were collected. The brains were fixed overnight in 4% PFA followed by a dehydration step with 30% sucrose in PBS. Brain samples were then frozen with optimum cutting temperature compound (O.C.T. compound; 4583, Sakura Finetek, CA, USA) and cut on a cryotome (Leica CM1850; Leica Biosystems, Wetzlar, Germany) into 30 μm thick coronal sections through the entire substantia nigra and striatum. All sections were made from the posterior end of the SN to the anterior end of the striatum. The sections were stored at – 20 ℃ in a cryoprotectant solution made with ethylene glycol, glycerol, and 0.2 M phosphate buffer.
Oct compound 4583
Manufactured by Sakura Finetek
Sourced in United States
OCT compound 4583 is a laboratory reagent used in optical coherence tomography (OCT) imaging. It is a critical component in OCT systems, enabling high-resolution, cross-sectional imaging of biological samples. The core function of this compound is to facilitate the optical interference necessary for OCT analysis. Further details on its intended use are not available.
Automatically generated - may contain errors
Lab products found in correlation
Cm1850, by Leica (1 mentions)
Mak194, by Merck Group (1 mentions)
Ht501128, by Merck Group (1 mentions)
Lidocaine hcl, by Merck Group (1 mentions)
A11122, by Thermo Fisher Scientific (1 mentions)
Sc 2780, by Santa Cruz Biotechnology (1 mentions)
Slowfade diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Nb100 737, by Novus Biologicals (1 mentions)
Goat anti rabbit igg tr, by Santa Cruz Biotechnology (1 mentions)
4 6 diamidino 2 phenylindole 2hcl dapi, by Merck Group (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Sucrose, by Merck Group (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
6 protocols using oct compound 4583
1
Comprehensive Tissue Collection for MPTP Murine Model
2
Lipid Droplet Staining in PFOA-Treated Mice
3
Comprehensive Livestock Metabolic Assessment
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Milk yield was recorded on 3 consecutive days at 0530 and 1500 h. Milk samples (50 mL) were collected at each milking from each cow on 3 consecutive days, and then stored at 4°C with preservative (1 mg/mL of potassium dichromate) until analyzed for milk composition by using an infrared analyzer with a 4-channel spectrophotometer (Sun et al., 2019; Foss MilkoScan, Foss Food Technology Co., Eden Prairie, MN) . Blood samples were collected between 0700 and 0900 h (before the morning feeding) from a coccygeal vein and immediately centrifuged at 3,500 × g for 15 min at 4°C. Serum was obtained and stored at -80°C until analysis. Liver tissue samples were taken from the 11th or 12th right intercostal space by liver puncture needle (Shanghai Surgical Equipment Factory, Shanghai, China) after blood collection. The intercostal space was shaved before the liver biopsy, sanitized with iodine scrub and 75% alcohol, and anesthetized with a subcutaneous injection of 2% lidocaine HCl (Sigma-Aldrich Co., St. Louis, MO). A scalpel blade was used to make a 3-mm stab incision in the skin. The puncture needle was then inserted through the intercostal muscle and into the liver. The liver tissue biopsies (~200 mg) were immediately frozen in liquid nitrogen or fixed with 4% paraformaldehyde or OCT compound (4583; Sakura Finetek Co., Torrance, CA).
4
Immunofluorescence Staining of Cryosectioned Tissues
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Colons were washed and fixed overnight at 4°C in a solution of 1% paraformadehyde in PBS. The tissues were incubated in a solution of 30% sucrose in PBS and the mixture of 30% sucrose and OCT compound 4583 (Sakura Finetek) separately at 4°C overnight. The samples were then embedded in OCT, frozen in a bath of ethanol cooled with liquid nitrogen and stocked at −80°C. Frozen samples were cut at 10-µm thickness and collected onto slides. Slides were dried at 50°C for 30 min and fixed in 1% paraformaldehyde for 10 min and processed for staining. The tissues were permeabilized in PBS/0.5% Triton X-100/0.3 M glycine at 37°C for 30 min and blocked in PBS/5% goat serum at room temperature for 1 h. The tissues were then incubated with indicated primary antibodies diluted (1:1,000) in PBS/5% goat serum at 4°C overnight (anti-green fluorescent PR, A11122 [Invitrogen]; and IL-22 antibody, NB100-737 [Novus]), and washed in PBS/0.2% Tween-20 at room temperature for 30 min three times. The tissues were incubated with Alexa dye-conjugated secondary antibodies (goat anti-rabbit IgG-TR, 1:200, sc-2780; Santa Cruz) and DAPI (1:200) in PBS/0.5% BSA at room temperature for 2 h and washed in PBS/0.2% Tween-20 at room temperature for 1 h five times before mounting with SlowFade Diamond Antifade Mountant (Life Technologies).
5
Immunofluorescence Tissue Staining Protocol
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Example 4
Tissue processing and staining procedures for immunofluorescence have been described previously (Peduto, L., et al., J. Immunol. 182, 5789-5799. (2009)). Briefly, tissues were fixed O/N at 4° C. in 4% paraformaldehyde (PFA) (Sigma), washed O/N in PBS, incubated in a solution of 30% sucrose (Sigma) until the samples sank, embedded in OCT compound 4583 (Sakura Finetek), frozen in a bath of isopentane cooled with liquid nitrogen and stocked at −80° C. Frozen blocs were cut at 8 μm thickness and sections were processed for staining: after blocking with 10% bovine serum in PBS containing 1% Triton (PBS-XG) for 1 hour at room temperature (RT), slides were incubated with primary antibodies (Abs) in PBS-XG overnight at 4° C., washed 3 times 5 min with PBS-XG, incubated with secondary conjugated Abs or streptavidin for 1 hour at RT, washed once, incubated with 4′6-diamidino-2-phenylindole-2HCl (DAPI) (Sigma) 5 min at RT, washed 3 times 5 min and mounted with Fluoromount-G (Southern Biotechnology Associates). Slides were examined with an Axiolmager M1 fluorescence microscope (Zeiss) equipped with a CCD camera and images were processed with AxioVision software (Zeiss). Mosaic images were generated using Spinning Disk Confocal microscopy (Cell Voyager) and images were analysed with ImageJ software.
6
Abdominal Aorta Perfusion for PFA Fixation
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To avoid chest pain from the surgical manipulation for PFA perfusion, we conducted the PFA perfusion via the abdominal aorta. A 22 G (for rats) or 30 G (for mice) needle was inserted into the abdominal aorta and perfused with 1% PFA in 0.1 M phosphate buffer (PB), followed by 4% PFA in 0.1 M PB. In some experiments, the rats were fixed transcardially to dissect out thoracic spinal cord and thoracic DRGs. The samples were post-fixed with 4% PFA in 0.1 M PB at 4 °C overnight and embedded using the O.C.T compound (4583; Sakura Finetek Japan Co., Tokyo, Japan) and frozen by powdered dry ice.
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