Immunocytochemistry experiment was performed as described previously (Noureddini et al., 2012). Briefly, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.05% Triton X-100. After blocking with 3% goat serum albumin, cells were incubated with primary antibodies for glial, neuronal and pre-neuronal markers at 37°C for 12 hours. The following primary antibodies and dilutions were used: mouse anti-β-tubulin-Tuj1 (1:500; Chemicon, Billerica, MA, USA); rabbit anti-glial fibrillary acidic protein (GFAP; 1:500; Sigma); rabbit anti-nestin (1:1,000; Sigma); mouse anti-microtubule-associated protein 2 (MAP-2; 1:500; Sigma). Then the cells were washed with PBS and reacted with the fluorescent isothiocyanate (FITC) conjugated secondary antibodies against rabbit and mouse Fc region (Sigma; 1:500) at room temperature for 2 hours. Finally, the cells were washed with PBS three times, and 4′,6-diamidino-2-phenylindole (DAPI) was used for DNA staining. The cells were visualized with Ceti immunofluorescence microscopy (Belgium).
Mouse anti microtubule associated protein 2 map 2
Manufactured by Merck Group
Sourced in United States
Mouse anti-microtubule-associated protein 2 (MAP-2) is a laboratory reagent used for the detection and analysis of microtubule-associated protein 2 in biological samples. MAP-2 is a structural protein found in neuronal cells and is involved in the stabilization and organization of the cytoskeleton.
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Lab products found in correlation
Rabbit anti gfap glial fibrillary acidic protein, by Merck Group (1 mentions)
Rabbit anti nestin, by Merck Group (1 mentions)
Biotinylated horse anti mouse antibody, by Vector Laboratories (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Mouse anti adenomatous polyposis coli apc, by Abcam (1 mentions)
Mouse anti neurofilament 200 nf200, by Merck Group (1 mentions)
Mouse anti glial fibrillary acidic protein, by Merck Group (1 mentions)
Penicillin and streptomycin, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by List Biological Laboratories (1 mentions)
Bio plex 200 system, by Bio-Rad (1 mentions)
Tnf α, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
70 μm cell strainer, by BD (1 mentions)
Cytotoxicity detection kit ldh, by Roche (1 mentions)
Alexa fluor 488 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Fv1200, by Olympus (1 mentions)
5 protocols using mouse anti microtubule associated protein 2 map 2
1
Immunocytochemical Analysis of Neural Cell Markers
2
Quantifying Myelin and Neuronal Integrity
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Coronal paraffin sections (8 µm) of paraformaldehyde (PFA)-fixed brains were incubated with mouse-anti-myelin basic protein (MBP) (Sternberger Monoclonals, Lutherville, MD,) or mouse-anti-microtubule-associated protein 2 (MAP2) (Sigma-Aldrich) followed by biotinylated horse-anti-mouse antibody (Vector Laboratories, Burliname, CA). Binding was visualized with Vectastain ABC kit (Vector Laboratories) and diaminobenzamidine. Ipsilateral MAP2 and MBP area loss was determined on sections corresponding to −1.85 mm from bregma in adult mouse brain. MBP and MAP2 staining were quantified using ImageJ software (http://rsb.info.nih.gov/ij/ ) and Adobe Photoshop CS5, respectively.
3
Immunofluorescence Staining of Spinal Cord
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Specific proteins were detected by using immunofluorescence staining (IFS) as reported previously [41 (link)]. Briefly, the post-fixed spinal cord was sectioned at 20-μm thickness with a cryostat. The sections were rinsed with 0.01 M phosphate-buffered saline (PBS) three times, blocked with 10 % goat serum for 30 min, and incubated with primary antibodies mixed in 0.3 % Triton X-100 overnight at 4 °C. After rinsing in PBS, the sections were incubated with secondary antibodies and examined under a fluorescence microscope. A summary of antibodies used is as follows: rabbit anti-β-tubulin III (Tju-1) (1:300, Sigma-Aldrich, St. Louis, MO, USA), mouse anti-microtubule-associated protein 2 (Map2) (1:1000, Sigma-Aldrich), mouse anti-glial fibrillary acidic protein (GFAP) (1:300, Sigma-Aldrich), mouse anti-neurofilament 200 (NF200) (1:300, Sigma-Aldrich), rabbit anti-myelin basic protein (MBP) (1:600, Chemicon, now part of Millipore Corporation, Billerica, MA, USA), and mouse anti-adenomatous polyposis coli (APC) (1:200, Abcam, Cambridge, UK).
4
Murine Cell Culture Assay Materials
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Cell culture materials (flasks, 24-well plates, tips) were obtained from Sarstedt Inc. (Newton, NC, USA), while 70-μm cell strainer was bought from BD (Franklin Lakes, NJ, USA). TNF-α, Dulbecco's modified Eagle's medium (DMEM), Hanks' buffered salt solutions (HBSS), antibiotics (penicillin and streptomycin), and RIPA buffer were supplied by Sigma-Aldrich (St. Louis, MO, USA). Heat-inactivated and certified fetal bovine serum (FBS) was bought from Invitrogen-GIBCO (Waltham, MA, USA). LPS was obtained from List Biological Laboratories, Inc. (#423; Campbell, CA, USA). Human recombinant galectin-3 was provided by Prof Anna Karlsson-Bengtsson, University of Gothenburg. The cytotoxicity detection kit (LDH) was bought from Roche Applied Science (Indianapolis, IN, USA) and BCA protein assay kit from Thermo Scientific Pierce (Waltham, MA, USA). Mouse IGF-1 and MCP-1 ELISA kits were obtained from R&D Systems (Minneapolis, MN, USA), while Bio-Plex ProTM Mouse cytokine Assay kit (M60-009 RDPD) and Bio-PlexTM200 system were provided by Bio-Rad Laboratories Inc. (Hercules, CA, USA). In addition, a SpectraMax ® Plus 384 Spectrophotometer from MD (Ramsey, MN, USA) was used. For staining histological sections, the following antibodies were used: mouse anti-microtubule-associated protein-2 (MAP-2) (Sigma-Aldrich, St. Louis, MO, USA) and mouse-anti-myelin basic protein (MBP) (SMI-94R; BioSite).
5
Immunostaining of Differentiated Cells
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Immunostaining was performed on day 7 (Fig. 2A). Differentiated cells were fixed with 4% paraformaldehyde for 15 min, and then treated with 0.1% Triton X-100 for 30 min. After incubation with 1% BSA-PBS for 30 min at room temperature, the cells were treated with primary antibodies [mouse anti-microtubule-associated protein 2 (MAP2; 1:200 dilution; Sigma-Aldrich)] overnight at 4C. After washing with PBS, the cells were incubated with secondary antibodies (Alexa Fluor 488 donkey anti-mouse IgG; 1:1,000 dilution; Invitrogen, Eugene, OR, USA) for 1 hr at room temperature. Cells were then treated with 2 g/mL of Hoechst 33342 solution for 15 min at room temperature. Immunofluorescence images were acquired using FV1200 (Olympus, Tokyo, Japan).
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