Neurons were fixed in 4% formaldehyde in PBS for 15 min, incubated in PBS + 10% TX-100 + 10% goat serum for 30 min, and labeled with rabbit anti-Nav1.6 antibody (Caldwell et al., 2000, 1:100), mouse monoclonal anti-ankyrinG (NeuroMab; 1:1000), or mouse monoclonal anti-MAP2 (Sigma, 1:1000) diluted in PBS containing 10% goat serum and 1% TX-100. Goat anti-rabbit or goat anti-mouse secondary antibodies conjugated to AlexaFluor-488, 594 or 647 (Molecular Probes, Life Technologies, Grand Island, NY, USA) were diluted 1:1000 in 10% goat serum and PBS.
Mouse monoclonal anti map2
Manufactured by Merck Group
Sourced in United States, Germany
The Mouse monoclonal anti-MAP2 is a laboratory reagent used for the detection and localization of Microtubule-Associated Protein 2 (MAP2) in various biological samples. MAP2 is a structural protein found primarily in the dendrites of mature neurons and is commonly used as a marker for neuronal cells.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Anti gfap, by Merck Group (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Mouse monoclonal anti nestin, by Merck Group (1 mentions)
Alexa fluor 555 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Axioskop, by Zeiss (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
4 protocols using mouse monoclonal anti map2
1
Immunolabeling Sodium Channel and Cytoskeletal Proteins
2
Quantifying Cell Death in Neuronal Cultures
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Prior to fixation of PNCs, SYTOX staining was performed to label dead cells. SYTOX stain (excitation/emission: 546/570 nm; ThermoFisher) diluted 1:5000 in live cell imaging solution was added to the cells and incubated for 15 min at RT. Cells were then fixed in 4% paraformaldehyde overnight as described [27 (link)]. The following antibodies were used: mouse monoclonal anti-MAP2 (1:200; Sigma) and donkey anti-mouse Alexa 488 (1:400; Invitrogen). Hoechst 33,258 stain (1:1000) was used to label nuclei.
Images were taken using confocal laser scanning microscopy (LSM 700, Zeiss) under 20X magnification. The number of SYTOX + and Hoechst + cells was assessed using ImageJ software (NIH, Bethesda, USA). The mean cell count from 2–4 different images per well per condition was calculated (3 wells per condition in Fig.1 C, 1 well per condition in Fig. 2 H). The ratio of SYTOX + cell numbers to the Hoechst + cell numbers in corresponding images was used to quantify the percentage of dead cells.
Images were taken using confocal laser scanning microscopy (LSM 700, Zeiss) under 20X magnification. The number of SYTOX + and Hoechst + cells was assessed using ImageJ software (NIH, Bethesda, USA). The mean cell count from 2–4 different images per well per condition was calculated (3 wells per condition in Fig.
3
Immunofluorescent Labeling of Neuronal and Glial Markers
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Microtubule associated protein-2 (MAP2) is a marker of mature neurons (Nakano et al., 2015; Razavi et al., 2015) and glial fibrillary acidic protein (GFAP) a marker of glial cells (Babaee et al., 2015; Pacey et al., 2015). Cells were washed twice with ice-cold phosphate buffered saline (PBS), fixed with 100% methanol for 7 minutes at −20°C, and permeated with fresh 4% paraformaldehyde for 20 minutes at room temperature. Cells were blocked with blocking buffer (10% goat serum in PBS containing 0.3% Triton X-100 and 0.03% NaN3) overnight at 4°C. Next, cells were incubated at 4°C for 24 hours with primary antibody diluted in blocking buffer, followed by incubation overnight at 4°C with secondary antibody diluted in blocking buffer. After washing with PBS, cells were stained with Hoechst 33342 (1:1,000; Pierce) for 30 minutes at room temperature and then viewed using a fluorescence microscope (Leica). The primary antibodies used were: mouse monoclonal anti-MAP2 (1:1,000; Millipore, Boston, MA, USA) and rabbit polyclonal anti-GFAP (1:500; Sigma). The secondary antibodies used were: Alexa Fluor 568-conjugated (red) goat anti-rabbit IgG (1:500; Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 488-conjugated (green) goat anti-mouse IgG (1:200; Invitrogen).
4
Immunofluorescence Analysis of Human iPSCs
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The human iPSCs cultured in TCP or PS inserts were fixed by adding 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 15 minutes and then permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 5 minutes. After blocking with 3% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) solution for 30 minutes, the cells were incubated with mouse anti-human primary antibodies (mouse monoclonal anti-neuronal β-III Tubulin, mouse monoclonal anti-Nestin and mouse monoclonal anti-MAP2 (all are from Millipore, Darmstadt, Germany)) at 4 °C overnight. After washing with PBS, the secondary antibodies (Alexa Fluor-488 goat anti-mouse IgG, 1:500; Alexa Fluor-555 goat anti-mouse IgG, 1:500; Life Technologies, Darmstadt, Germany) were added and incubated for 60 minutes. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA).
After 3 times of washing with PBS, the samples were visualized using a fluorescence microscope (AxioSkop, Carl Zeiss, Jena, Germany) or scanned with a confocal laser scanning microscope (LSM 780, Carl Zeiss, Jena, Germany).
For the cell characterization staining, the human iPSCs were stained with the Fluorescent Human ES/iPS Cell Characterization Kit (Millipore, Darmstadt, Germany) following the manufacturer's protocol.
After 3 times of washing with PBS, the samples were visualized using a fluorescence microscope (AxioSkop, Carl Zeiss, Jena, Germany) or scanned with a confocal laser scanning microscope (LSM 780, Carl Zeiss, Jena, Germany).
For the cell characterization staining, the human iPSCs were stained with the Fluorescent Human ES/iPS Cell Characterization Kit (Millipore, Darmstadt, Germany) following the manufacturer's protocol.
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