mouse anti-map2 antibody smi-52  (Covance)

Journal: Neuroscience research

Article Title: Parallel high throughput neuronal toxicity assays demonstrate uncoupling between loss of mitochondrial membrane potential and neuronal damage in a model of HIV-induced neurodegeneration

doi: 10.1016/j.neures.2011.01.013

Figure Lengend Snippet: (a) MAP2 RMF in the linear range of fluorescence is maximal (Brewer, 1995) compared with no primary control background when 0.5μg of Fluorocillin substrate is incubated for 60 min. (b) Background-subtracted MAP2 RMF values increase linearly with increased plating density of primary rat cortical neurons sevaluated after 3 days in culture. n=2-3 wells (a), n=14-16 wells (b) one-way ANOVA, Newman-Keuls, p<0.05 for all differences in cell densities ≥10×103; in some cases the SEM was smaller than the plotting symbol.

Article Snippet: After blocking for 1 h with 5% normal goat serum in phosphate buffered saline (PBS), plates were incubated with mouse anti-MAP2 antibody (SMI-52, 1:1000, Covance, Berkley, CA) diluted in Normal Antibody Diluent (ScyTek Labs, Logan, UT, USA) overnight at 4°C, followed by washing with PBS with 0.1% Tween-20 (PBS-T).

Techniques: Fluorescence, Control, Incubation

Journal: Neuroscience research

Article Title: Parallel high throughput neuronal toxicity assays demonstrate uncoupling between loss of mitochondrial membrane potential and neuronal damage in a model of HIV-induced neurodegeneration

doi: 10.1016/j.neures.2011.01.013

Figure Lengend Snippet: (a) Cultures were labeled for MAP2 (red) and DAPI (blue) and imaged by immunofluorescent laser confocal microscopy at various timepoints (2, 4, and 20 hr) following the application of HIV- or Mock-MDM (1:10 dilutions). In HIV-MDM-treated cultures, dendritic beading is evident by 2hr, and widespread loss of MAP2-positive neurons and dendrites, as well as condensed nuclei, is pronounced by 20h. (b) 96-well plate cortical cultures were treated with serial dilutions of HIV-MDM for 40 hr and were assayed for changes in MAP2-positive neurons by MAP2 CB-ELISA (left) or by hand-counting MAP2-positive cells (right). Each dilution of HIV-MDM resulted in a significant decrease in % of MAP2 RMF (left) or % of neuron number (right) when compared with untreated. Each dilution of HIV-MDM was also significantly different from all other HIV-MDM treatments evaluated by the same assay (p<0.001 one-way ANOVA, Newman-Keuls). For any particular dilution of HIV-MDM, there was no statistical difference in the % changes from untreated obtained from the MAP-CB ELISA (MAP2 RMF values) compared with the % change from untreated obtained from traditional hand-counting assay (number of MAP-2 positive cells) in the same plate (p<0.05, one-way ANOVA, Bonferroni), n=6-12 wells. (c) 96-well plate cortical cultures were treated with serial dilutions of glutamate for 24 hr. Values are shown normalized to vehicle control. Each increase concentration of glutamate resulted in a statistically significant (p<0.001, one-way ANOVA, Newman-Keuls) observed increase in MAP2 RMF compared with all other dilutions, with two exceptions: for 1μM glutamate compared with vehicle p<0.05, and 10μM compared with 5μM is not statistically different.

Article Snippet: After blocking for 1 h with 5% normal goat serum in phosphate buffered saline (PBS), plates were incubated with mouse anti-MAP2 antibody (SMI-52, 1:1000, Covance, Berkley, CA) diluted in Normal Antibody Diluent (ScyTek Labs, Logan, UT, USA) overnight at 4°C, followed by washing with PBS with 0.1% Tween-20 (PBS-T).

Techniques: Labeling, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Control, Concentration Assay

Journal: Neuroscience research

Article Title: Parallel high throughput neuronal toxicity assays demonstrate uncoupling between loss of mitochondrial membrane potential and neuronal damage in a model of HIV-induced neurodegeneration

doi: 10.1016/j.neures.2011.01.013

Figure Lengend Snippet: Loss of MAP2 RMF as measured by MAP2 CB-ELISA in 2-week old cortical cultures either untreated or following treatment with 1:10 or 1:40 dilutions of HIV-MDM at 3, 6, 12, 24, 36, and 60 hr. By 12 hr, both dilutions of HIV-MDM showed significant loss of MAP2 RMF compared with untreated cultures. MAP2 RMF loss following treatment with a 1:40 dilution of HIV-MDM was significantly reversed at 60 hr compared with 36 hr. n=12-16 wells; one-way ANOVA, Newman-Keuls, * p<0.01 compared with 36 hr; in some cases the SEM was smaller than the plotting symbol.

Article Snippet: After blocking for 1 h with 5% normal goat serum in phosphate buffered saline (PBS), plates were incubated with mouse anti-MAP2 antibody (SMI-52, 1:1000, Covance, Berkley, CA) diluted in Normal Antibody Diluent (ScyTek Labs, Logan, UT, USA) overnight at 4°C, followed by washing with PBS with 0.1% Tween-20 (PBS-T).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Neuroscience research

Article Title: Parallel high throughput neuronal toxicity assays demonstrate uncoupling between loss of mitochondrial membrane potential and neuronal damage in a model of HIV-induced neurodegeneration

doi: 10.1016/j.neures.2011.01.013

Figure Lengend Snippet: (a) 5-week-old cultures were left unexposed or exposed for 20 hr to 1:40 or 1:10 dilutions of HIV- or Mock-MDM with or without treatment with 20μM qVD-OPH, 10μM MDL28170, or 10μM MK801. There were no differences between any conditions in cultures exposed to Mock-MDM. At both dilutions of HIV-MDM, MDL28170 and MK801 significantly reduced the loss of MAP2 RMF. Cultures treated with qVD-OPH did not show statistical differences between treatments. ^p<0.05, *p<0.001 compared with vehicle-treated cultures receiving matching dilution of HIV-MDM (1:10 – black bars; 1:40 – hatched bars), or the vehicle-treated control cultures not receiving HIV-MDM (white bar) compared with vehicle-treated cultures receiving either 1:10 or 1:40 dilutions of HIV-MDM, one-way ANOVA, Newman-Keuls, n=13-30 wells. (b) MAP2 RMF loss in cultures exposed to a 1:10 dilution of HIV-MDM for 24 hr was not reduced when treated with various concentrations of qVD-OPH compared with vehicle-treated (0.5% DMSO) cultures. (c) In a separate experiment, cultures receiving a 1:10 dilution of HIV-MDM for 25 hr had reduced loss of MAP2 RMF when they were treated with 1, 10, and 100 μM MDL28170 compared with vehicle (1% DMSO) treated cultures. For b and c, v=vehicle; ^p<0.05, *p<0.001 compared with vehicle in HIV-MDM-treated cultures, one-way ANOVA, Dunnett’s, n=6-24 wells. All values are presented as a percentage of vehicle-treated control cultures (dash line, open circle). Maximum MAP2 RMF loss is indicated with a dashed line with closed circle. In some cases the SEM was smaller than the plotting symbol.

Article Snippet: After blocking for 1 h with 5% normal goat serum in phosphate buffered saline (PBS), plates were incubated with mouse anti-MAP2 antibody (SMI-52, 1:1000, Covance, Berkley, CA) diluted in Normal Antibody Diluent (ScyTek Labs, Logan, UT, USA) overnight at 4°C, followed by washing with PBS with 0.1% Tween-20 (PBS-T).

Techniques: Control

Journal: Neuroscience research

Article Title: Parallel high throughput neuronal toxicity assays demonstrate uncoupling between loss of mitochondrial membrane potential and neuronal damage in a model of HIV-induced neurodegeneration

doi: 10.1016/j.neures.2011.01.013

Figure Lengend Snippet: Cortical cultures exposed to a 1:20 dilution of HIV-MDM showed decreased TMRM fluorescence (open symbols and dotted lines) by 15 min and decreased MAP2 RMF by 90 min (closed symbols and solid lines). Cccp+oligomycin was used to uncouple mitochondrial electron chain transport and thoroughly depolarize cultures in order to assess background TMRM fluorescence (2.34±0.90) and is indicated by a dotted line. For visual purposes, untreated control cultures are placed near the 0 min time point at slightly offset (−0.8, 0, and 0.8) x-axis positions in order to avoid symbol overlap. ^p<0.05, *p<0.001 compared with untreated, one-way ANOVA, Dunnett’s; †p<0.01, #p<0.001 compared with Mock-MDM at the same time point, unpaired two-tailed t-test, n=6-12 wells; in some cases the SEM was smaller than the plotting symbol.

Article Snippet: After blocking for 1 h with 5% normal goat serum in phosphate buffered saline (PBS), plates were incubated with mouse anti-MAP2 antibody (SMI-52, 1:1000, Covance, Berkley, CA) diluted in Normal Antibody Diluent (ScyTek Labs, Logan, UT, USA) overnight at 4°C, followed by washing with PBS with 0.1% Tween-20 (PBS-T).

Techniques: Fluorescence, Control, Two Tailed Test

Journal: Neuroscience research

Article Title: Parallel high throughput neuronal toxicity assays demonstrate uncoupling between loss of mitochondrial membrane potential and neuronal damage in a model of HIV-induced neurodegeneration

doi: 10.1016/j.neures.2011.01.013

Figure Lengend Snippet: Two-week-old cortical cultures were exposed to a 1:20 dilution of HIV- or Mock-MDM. a) Neuronal damage, as determined by loss of MAP2 RMF by the MAP2 CB-ELISA and by automated image analysis expressed as integrated pixel intensity of MAP2, is significant by 4 hr and is prevented by MDL28170. In addition, exclusion of PI by cells in the cultures is decreased at 4 hr and is also prevented by MDL28170. b) Wells used in the MAP2 assay were reused for immunofluorescent imaging. Images in the left column show MAP2 (green), GFAP (red), and DAPI (blue). Images on the right column show PI labeling (red) in the same field. PI was added prior to fixation of the cultures in order to label only cells with compromised membranes. Little damage is seen by 1 hr (not shown), but by 4 hr loss of MAP2 and increased PI labeling are evident. c) MAP2 loss (solid symbols, solid lines) is detectable prior to loss of PI-exclusion (open symbols, dotted lines) following treatment with HIV-MDM. d) MDL28170 fails to prevent loss of TMRM fluorescence following HIV- or Mock-MDM treatment at 1 or 4 hr. Cccp+oligomycin was used to depolarize cultures in order to assess background TMRM fluorescence (4.43±0.82) and is indicated by a dotted line. ^p<0.05, †p<0.01, *p<0.001 compared with assay specific b) HIV-MDM treated or c) untreated cultures, one-way ANOVA, Newman-Keuls, n=4-16 wells.

Article Snippet: After blocking for 1 h with 5% normal goat serum in phosphate buffered saline (PBS), plates were incubated with mouse anti-MAP2 antibody (SMI-52, 1:1000, Covance, Berkley, CA) diluted in Normal Antibody Diluent (ScyTek Labs, Logan, UT, USA) overnight at 4°C, followed by washing with PBS with 0.1% Tween-20 (PBS-T).

Techniques: Enzyme-linked Immunosorbent Assay, Imaging, Labeling, Fluorescence