P2287 100ml

Manufactured by Merck Group

P2287-100ML is a laboratory reagent produced by Merck Group. It is a 100 milliliter solution intended for use in scientific research and analysis. The core function of this product is to serve as a laboratory reagent, without further interpretation of its intended applications.

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Lab products found in correlation

Bovine serum albumin (bsa), by Serva Electrophoresis (1 mentions) Toluol, by Merck Group (1 mentions) Rabbit anti iba1, by Fujifilm (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Anti mouse irdye 680rd, by LI COR (1 mentions) Anti rabbit irdye 800cw, by LI COR (1 mentions) Odyssey infrared imager, by LI COR (1 mentions) Precast gel, by Bio-Rad (1 mentions) Ab190565, by Abcam (1 mentions) Ab196175, by Abcam (1 mentions) Ab7481, by Merck Group (1 mentions) Lsm 800, by Zeiss (1 mentions)

3 protocols using p2287 100ml

Immunohistochemistry of Microglia in Brain Slices

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Immunohistochemistry was performed with free-floating sections. Brain slices were washed once with PBS containing 0.05% Tween20 (PBS-T) (P2287-100ML; Sigma-Aldrich) and treated with 60% methanol (VWR Prolabo) for 1 hour. Sections were incubated in blocking solution consisting of PBS-T plus 2% bovine serum albumin (11930; Serva), 0.3% milk powder (T145; Roth), and 0.5% donkey normal serum (017-000-001; Jackson Immuno Research) for 30 min prior to incubation with the primary antibody. Immunofluorescence staining for microglia was performed with rabbit anti-Iba-1 (1:500; WAKO) in blocking solution overnight at 4 °C, followed by secondary antibody donkey-anti-rabbit Cy3 (1:250, 711-165-152; Dianova) for 1 hour. Finally, slices were washed three times and incubated for 10 min with DAPI (1:10,000; Sigma) at 4 °C and washed once. For immigrated GFP⁺ MSCs, no immunohistochemical enhancement was used. Slices were mounted on glass slides, dried, and coverslipped with entellan in toluol (108323; Merck).

Fabian C., Naaldijk Y., Leovsky C., Johnson A.A., Rudolph L., Jaeger C., Arnold K, & Stolzing A. (2017). Distribution pattern following systemic mesenchymal stem cell injection depends on the age of the recipient and neuronal health. Stem Cell Research & Therapy, 8, 85.

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Western Blot Protein Detection

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Precast gels were purchased from BioRad and were run for 110 min at 100 V. 1× running buffer was composed of 3.0 g Tris base, 14.4 g glycine and 1.0 g SDS in 1 l water. Gels were transferred to nitrocellulose for 1 h at 100 V in 1× transfer buffer. 10× transfer buffer was composed of 30.0 g Tris and 144.0 g glycine in 1 l water. 1× Transfer buffer is composed of 10% 10× running buffer, 20% methanol, and 70% water. Following transfer, blots were blocked with 5% bovine serum albumin (BSA) (Sigma: A9647-100G) in TBS with 0.05% Tween-20 (TBST) (Sigma: P2287-100ML) for 1 h. Antibodies were made with 5% BSA in TBST. Primary antibodies: GAPDH 1:5000 mouse (Thermofisher MA5-15738). GFP 1:1000 rabbit (Invitrogen A11122). Secondary antibodies: IRDye 680RD anti-mouse 1:20,000 (Licor 926-68170). IRDye 800CW anti-rabbit 1:20,000 (Licor 925-68024). Primary antibodies were incubated overnight at 4 °C. Following three washes with TBST of at least 10 min each, secondary antibodies were incubated for 1 h at room temperature. Three washes with TBST of at least 10 min each were sufficient to remove the secondary antibodies. Membranes were imaged using a Licor Odyssey infrared imager.

Lobas M.A., Tao R., Nagai J., Kronschläger M.T., Borden P.M., Marvin J.S., Looger L.L, & Khakh B.S. (2019). A genetically encoded single-wavelength sensor for imaging cytosolic and cell surface ATP. Nature Communications, 10, 711.

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Immunofluorescence Analysis of Tight Junctions in BBB

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A piece of the membrane containing cells on both sides was incubated in a blocking buffer comprising 5% goat serum (Abcam, #ab7481), 0.1% TritonX 100 (Sigma, # 9036-19-5), and 1% BSA (MP, #180561) for 2 hours. Cells were washed in PBS with 0.1% Tween-20 (Sigma, #P2287-100ML). Conjugated antibodies for claudin 5 (Invitrogen, #362588) at 1:200 and ZO-1 (Invitrogen, #MA3-39100-A647) at 1:100 dilution in PBS with 0.1% Tween-20 were added to the samples and incubated overnight at 4°C. A separate piece of the same membrane was used for cell identification by staining it with cell marker conjugated-antibodies, anti-S1OO beta for astrocytes (Abcam, #ab196175), anti-CD146 (Abcam, #ab196448) for pericytes and hBMECs, and anti-NeuN antibody (Abcam, ab190565) for neurons, with all Abs at 1:200 dilution. The following day, samples were washed twice with PBS and 0.1% Tween-20. After washing, samples were fixed with 4% paraformaldehyde for 10 minutes in the dark. The fixed samples were washed with PBS and air-dried on a glass cover slip. Samples were analyzed for expression of tight junction proteins using a laser scanning microscope (Zeiss LSM800). Of note, no quantification of fluorescence intensities for proteins was performed and comparison of fluorescence was assessed visually.

Malik J.R., Fletcher C.V., Podany A.T., Dyavar S.R., Scarsi K.K., Pais G.M., Scheetz M.H, & Avedissian S.N. (2023). A novel 4-cell in-vitro blood-brain barrier model and its characterization by confocal microscopy and TEER measurement. Journal of neuroscience methods, 392, 109867.

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