After eight days of cells cultured in NGF(+) or NGF(−) Differentiation Medium on different scaffolds, the immunostaining of cytoskeletal proteins -Tubulin was carried out to observe the cell phenotype and neurite extension of differentiated PC12 cells. The reason for choosing α-Tubulin as the staining marker was based on former studies [28 (link),29 (link)]. All the scaffolds were rinsed with PBS, fixed in neutral buffer formalin solution (Sigma) for 20 min and permeabilized with 0.25% Triton X-100. The nonspecific binding was blocked by incubating with 3% BSA in PBS for 90 min. Subsequently the samples were incubated with a neuronal-specific Mouse anti- -Tubulin antibody (Invitrogen, Camarillo, CA, USA), followed by the staining of Alexa Fluor 594 Goat Anti-Mouse IgG (H + L) antibody (Invitrogen), and the nuclei was stained with DAPI (Thermo Scientific). The immunostained samples were mounted on glass slides and fluorescent images were taken using a laser scanning confocal microscope (Zeiss LSM700). Some samples incubated with PBS instead of anti-α-Tubulin antibody were used as the blank control group.
Formalin neutral buffer solution
Manufactured by Merck Group
Sourced in Germany, United States
Formalin neutral buffer solution is a laboratory reagent used for the fixation and preservation of biological samples. It is a mixture of formaldehyde and a buffered saline solution, designed to maintain a neutral pH. The primary function of this product is to prevent tissue degradation and preserve the sample's structural and molecular integrity for downstream analysis.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 goat anti mouse igg h l antibody, by Thermo Fisher Scientific (1 mentions)
Mouse anti α tubulin antibody, by Thermo Fisher Scientific (1 mentions)
Dako envision flex kit, by Agilent Technologies (1 mentions)
S 100 antibody, by Merck Group (1 mentions)
Cx21 light microscope, by Olympus (1 mentions)
5 protocols using formalin neutral buffer solution
1
Immunostaining of Differentiated PC12 Cells
2
Immunohistochemical Analysis of Nerve Grafts
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Grafts were fixed in 10% v/v neutral buffer formalin solution (Sigma-Aldrich, Darmstadt, Germany) paraffin embedded and sectioned. Then, the slides were deparafinnized, rehydrated and blocked. Dako Envision Flex kit was used for the immunohistochemistry assay according to manufacturer’s instructions (Dako, Agilent, Glostrup, Denmark). Briefly, nerve graft sections were incubated at 4 °C over night with rabbit anti-neurofilament 200 (nf 200) antibody (1:80, Sigma, St. Louis, MO, USA) to identify axons and S100 antibody (1:100, Sigma, St. Louis, MO, USA) to identify Schwann cells. Briefly, washes were performed, and addition of horseradish peroxidase (HRP) conjugated with goat secondary antibody against rabbit and mouse was performed. The slides were incubated at Room Temperature (RT) for 45 min. Finally, 3′3 diaminobenzidine (DAB) was added to the slides. Slides were visualized by light microscopy and images were acquired with ΙC Capture 2.2 software and processed with imageJ software version 1.52g.
3
Odontoclast Identification by TRAP Staining
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Cultures were fixed in 10% formalin neutral buffer solution (Sigma-Aldrich, St. Louis, MO, USA). After serial washing with PBS three times, odontoclasts were detected by staining for tartrate-resistant acid phosphatase (TRAP), a marker enzyme for odontoclasts, using a kit following the manufacturer's recommendations (Cosmo Bio Co., Ltd., Tokyo, Japan). TRAP activity was observed under an optical microscope with 63x magnification (Axio Vert. A1, Zeiss, Göttingen, Germany) and TRAP-positive cells were counted.
4
Histological Analysis of Hairless Mouse Skin
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Hairless mouse dorsal skin tissue was collected, fixed with 10% formalin neutral buffer solution (Sigma-Aldrich), embedded in paraffin, and cut into 4 μm-thick packets, deparaffinized with xylene, and rehydrated via graded alcohol. Hematoxylin eosin (HE) staining is used for histological observations of skin structure, thickness of the epidermis and dermis. HE staining was carried out in several stages starting with deparaffinization, hydration, hematoxylin staining, eosin staining, and finally dehydration. The preparations were analyzed at 10 random locations per slide using an Olympus Cx21 light microscope with 1000x magnification. Each specimen is photographed under a camera with 48MP. Histological changes and collagen fiber density were evaluated and measured using ImageJ 1.53e software.
5
Synucleinopathy Induction in Transgenic Mice
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Six- to eight-week-old FVB;129S6-Sncatm1NbmTg(SNCA)1Nbm/J mice (short: Tg(SNCA)1Nbm/J mice, The Jackson Laboratory) were anaesthetized with isoflurane and stereotaxically injected with 30 μl of cortical brain extract from MSA or probable iLBD cases or PBS into the left striatum (coordinates: +0.2 mm relative to the bregma, +2.0 mm relative to the midline, 2.6 mm below the dura) using a 27-gauge disposable hypodermic syringe. After recovery from surgery, animals were monitored daily for health and three times weekly for signs of neurologic illness such as reduced grooming, ataxia, tremor, bradykinesia, akinesia, lethargy, circling, tail rigidity, paraparesis, paralysis, kyphosis, and more. At 3, 6, and 9 months post injection the mice were sacrificed by overdose with ketamine/xylazine and then transcardially perfused with 0.9 % saline followed by 10 % formalin neutral buffer solution (Sigma). Brains were fixed overnight with 1 % formalin neutral buffer solution for immunohistochemistry. For biochemical analysis, brains were snap-frozen on dry ice and stored at −80 °C. All animal studies were approved by the animal protection committee of the North Rhine-Westphalia State Environment Agency (LANUV).
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