Journal: Cellular and Molecular Neurobiology

Article Title: Laser-Induced Axotomy of Human iPSC-Derived and Murine Primary Neurons Decreases Somatic Tau and AT8 Tau Phosphorylation: A Single-Cell Approach to Study Effects of Acute Axonal Damage

doi: 10.1007/s10571-023-01359-z

Figure Lengend Snippet: Somatic total Tau and phospho-Tau levels are not elevated after induced axon lesion in human iPSC-derived neurons independent of regrowth activity. Human iPSC-derived neurons were fixed after axotomy and subsequent monitoring at different time points (see methods for details), and immunostained with a polyclonal anti-panTau antibody (K9JA) and a monoclonal antibody recognizing Tau phosphorylation (AT8 epitope). a and b : Representative images of two iPSC-derived neurons that were grown, fixed, and immunostained in the same chamber, either after axotomy ( a ) or without axotomy ( b ) after 3 h monitoring. The left panels show the immunostained neurons before axotomy. The dotted violet lines indicate the location of axotomy. Scale bars (refer to all panels in b and d ): 20 µm. c and d : Quantification of the total Tau ( c ) and phospho-Tau ( d ) signal intensity in the somata of axotomized neurons, normalized to the signals of non-axotomized neurons. e : Ratio of phospho-Tau and total Tau levels in the somata of axotomized neurons, normalized to the ratio of non-axotomized neurons. f and g : Total Tau ( f ) and phospho-Tau levels ( g ) in axotomized neurons depending on the regrowth after axotomy. Grey dots represent individual neurons, colored bars indicate the arithmetic mean of all neurons, error bars represent the standard deviation (SD). An ordinary two-way ANOVA with Sidak’s multiple comparison test was performed for the determination of significant differences. Exact adjusted p-values are given for all comparisons. For detailed test statistics: see Supplemental Material 1

Article Snippet: The following primary antibodies were used: rabbit anti-TAU (K9JA) (1:1000, #A0024, DAKO), mouse anti-phospho Tau (AT8) (1:1000, #MN1020, ThermoFisher Scientific).

Techniques: Derivative Assay, Activity Assay, Standard Deviation, Comparison

Journal: Cellular and Molecular Neurobiology

Article Title: Laser-Induced Axotomy of Human iPSC-Derived and Murine Primary Neurons Decreases Somatic Tau and AT8 Tau Phosphorylation: A Single-Cell Approach to Study Effects of Acute Axonal Damage

doi: 10.1007/s10571-023-01359-z

Figure Lengend Snippet: Somatic total Tau and phospho-Tau levels are not elevated after induced axon lesion in mouse primary forebrain neurons. Mouse primary forebrain neurons were fixed after axotomy and subsequent monitoring at different time points (see methods for details), and immunostained with a polyclonal anti-panTau antibody (K9JA) and a monoclonal antibody recognizing Tau phosphorylation (AT8 epitope). a & b : Representative images of two mouse primary neurons that were grown, fixed, and immunostained in the same chamber, either after axotomy ( a ) or without axotomy ( b ) after 3 h monitoring. The left panels show the immunostained neurons before axotomy. The dotted violet lines indicate the location of axotomy. Scale bars (refer to all panels in b&d): 20 µm. c and d : Quantification of the total Tau ( c ) and phospho-Tau ( d ) signal intensity in the somata of axotomized neurons, normalized to the signals of non-axotomized neurons. e : Ratio of phospho-Tau and total Tau levels in the somata of axotomized neurons, normalized to the ratio of non-axotomized neurons. f and g : Total Tau ( f ) and phospho-Tau levels ( g ) in axotomized neurons depending on the regrowth after axotomy. Grey dots represent individual neurons, colored bars indicate the arithmetic mean of all neurons, error bars represent the standard deviation (SD). An ordinary two-way ANOVA with Sidak’s multiple comparison test was performed for the determination of significant differences. Exact adjusted p -values are given for all comparisons. For detailed test statistics: see Supplemental Material 1

Article Snippet: The following primary antibodies were used: rabbit anti-TAU (K9JA) (1:1000, #A0024, DAKO), mouse anti-phospho Tau (AT8) (1:1000, #MN1020, ThermoFisher Scientific).

Techniques: Standard Deviation, Comparison

Journal: The Journal of Neuroscience

Article Title: C-Terminally Truncated Forms of Tau, But Not Full-Length Tau or Its C-Terminal Fragments, Are Released from Neurons Independently of Cell Death

doi: 10.1523/JNEUROSCI.0387-15.2015

Figure Lengend Snippet: Antibodies used, their source, and working concentrations

Article Snippet: All tissue culture reagents were obtained from Invitrogen and, where appropriate, the medium used to condition cells was centrifuged (100,000 × g for 18 h and 4°C) to remove exosomes ( Théry et al., 2001 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Antibody Source Concentration Alix 3A9 Cell Signaling Technology 1 μg/ml Flotillin-1 18/Flotillin-1 BD Biosciences 1 μg/ml HIV glycoprotein 120 46-4 ATCC 1 μg/ml PrP ICSM35 D-Gen 0.5 μg/ml β3-tubulin ab68193 Abcam 1:250 dilution β3-tubulin TUJ1 Covance 5 μg/ml Tau (aa 6-18) Tau12 (human) EMD Millipore 0.1 μg/ml a Tau (aa 7-13) HJ9.4 (rodent) Gift from David Holtzman 1 μg/ml a Tau (aa 194-198) BT2 (human + rodent) Thermo Scientific 1 μg/ml a , b Tau ( aa 210-241) Tau5 (human + rodent) Cell Signaling Technology 1 μg/ml c Tau (aa 243-441) K9JA (human + rodent) DAKO 1 μg/ml a , b , c Tau (aa 404-441) Tau46 (human + rodent) Cell Signaling Technology 1 μg/ml a Misfolded tau (residues 7-9 + 313-322) MC1 Gift from Peter Davies 5 μg/ml a Open in a separate window Concentrations used for Western blotting: a 5 μg/ml for IP, b 2.5 μg/ml for ELISA, c 5 μg/ml for ELISA.

Techniques: Concentration Assay

Journal: The Journal of Neuroscience

Article Title: C-Terminally Truncated Forms of Tau, But Not Full-Length Tau or Its C-Terminal Fragments, Are Released from Neurons Independently of Cell Death

doi: 10.1523/JNEUROSCI.0387-15.2015

Figure Lengend Snippet: Characterization of β3T and tau ELISAs. A, Calibration curve of β3T standard. The ELISA displays an LLOQ of ∼125 pg/ml. B, The BT2-Tau5 ELISA detects FL rodent adult tau 430 (■) and human tau 441 (●) with an LLOQ of ∼30 pg/ml, but does not detect K19. C, K9JA-K9JA ELISA detects FL rodent adult tau (■), human tau 441 (●), and K19 (▴) with an LLOQ of ∼30 pg/ml. D, K9JA-Tau46 ELISA detects FL rodent adult tau (■) and human tau 441 (●) with an LLOQ of ∼125 pg/ml, but does not detect K19. E, HJ9.4-Tau46 ELISA detects FL rodent adult tau (■) and the Tau12-Tau46 ELISA detects human tau 441 (●). In each case, the LLOQ is 125 pg/ml. Each data point is the average ± SD of three replicates. Where error bars are not visible, the SD is smaller than the size of the symbol. See Figure 3 for the location of mAb epitopes and the tau species predicted to be detected by each assay.

Article Snippet: All tissue culture reagents were obtained from Invitrogen and, where appropriate, the medium used to condition cells was centrifuged (100,000 × g for 18 h and 4°C) to remove exosomes ( Théry et al., 2001 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Antibody Source Concentration Alix 3A9 Cell Signaling Technology 1 μg/ml Flotillin-1 18/Flotillin-1 BD Biosciences 1 μg/ml HIV glycoprotein 120 46-4 ATCC 1 μg/ml PrP ICSM35 D-Gen 0.5 μg/ml β3-tubulin ab68193 Abcam 1:250 dilution β3-tubulin TUJ1 Covance 5 μg/ml Tau (aa 6-18) Tau12 (human) EMD Millipore 0.1 μg/ml a Tau (aa 7-13) HJ9.4 (rodent) Gift from David Holtzman 1 μg/ml a Tau (aa 194-198) BT2 (human + rodent) Thermo Scientific 1 μg/ml a , b Tau ( aa 210-241) Tau5 (human + rodent) Cell Signaling Technology 1 μg/ml c Tau (aa 243-441) K9JA (human + rodent) DAKO 1 μg/ml a , b , c Tau (aa 404-441) Tau46 (human + rodent) Cell Signaling Technology 1 μg/ml a Misfolded tau (residues 7-9 + 313-322) MC1 Gift from Peter Davies 5 μg/ml a Open in a separate window Concentrations used for Western blotting: a 5 μg/ml for IP, b 2.5 μg/ml for ELISA, c 5 μg/ml for ELISA.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: The Journal of Neuroscience

Article Title: C-Terminally Truncated Forms of Tau, But Not Full-Length Tau or Its C-Terminal Fragments, Are Released from Neurons Independently of Cell Death

doi: 10.1523/JNEUROSCI.0387-15.2015

Figure Lengend Snippet: Healthy N2a cultures express low levels of tau, some of which can be detected in conditioned medium. A, Bright-field image of N2a cells after 24 h of conditioning. Scale bar, 50 μm. Because the cells were almost confluent and it was difficult to distinguish one cell from another, an edge of lower density is shown. B, Western blot analysis of lysates (12.5 μg of total protein/lane) treated with or without calf intestine alkaline phosphatase (CIP) using anti-tau antibodies HJ9.4 (aa 7–13), K9JA (aa 243–441), Tau46 (aa 404–441), and the negative control antibody 46–4. Recombinant rodent tau 431 (Rec), 10 ng, and CIP-treated mouse brain extract (Mse), 2.5 μg of total proteins, were loaded as controls and tau bands are indicated with a bracket. CIP treatment was used as an additional control because dephosphorylated tau tends to migrate a little faster. C, Medium collected from N2a cultures after 6, 24, and 48 h was analyzed for the presence of tau, LDH, and β3T. Percentage release was calculated according to the following equation: ([analyte in CM])/([analyte in CM] + [analyte in lysate]) × 100. D, Summary of five separate N2a-conditioning experiments reveals that tau is detected in CM at levels disproportionate to cytoplasmic marker proteins. Note that no β3T was detected in medium conditioned for 24 h.

Article Snippet: All tissue culture reagents were obtained from Invitrogen and, where appropriate, the medium used to condition cells was centrifuged (100,000 × g for 18 h and 4°C) to remove exosomes ( Théry et al., 2001 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Antibody Source Concentration Alix 3A9 Cell Signaling Technology 1 μg/ml Flotillin-1 18/Flotillin-1 BD Biosciences 1 μg/ml HIV glycoprotein 120 46-4 ATCC 1 μg/ml PrP ICSM35 D-Gen 0.5 μg/ml β3-tubulin ab68193 Abcam 1:250 dilution β3-tubulin TUJ1 Covance 5 μg/ml Tau (aa 6-18) Tau12 (human) EMD Millipore 0.1 μg/ml a Tau (aa 7-13) HJ9.4 (rodent) Gift from David Holtzman 1 μg/ml a Tau (aa 194-198) BT2 (human + rodent) Thermo Scientific 1 μg/ml a , b Tau ( aa 210-241) Tau5 (human + rodent) Cell Signaling Technology 1 μg/ml c Tau (aa 243-441) K9JA (human + rodent) DAKO 1 μg/ml a , b , c Tau (aa 404-441) Tau46 (human + rodent) Cell Signaling Technology 1 μg/ml a Misfolded tau (residues 7-9 + 313-322) MC1 Gift from Peter Davies 5 μg/ml a Open in a separate window Concentrations used for Western blotting: a 5 μg/ml for IP, b 2.5 μg/ml for ELISA, c 5 μg/ml for ELISA.

Techniques: Western Blot, Negative Control, Recombinant, Marker

Journal: The Journal of Neuroscience

Article Title: C-Terminally Truncated Forms of Tau, But Not Full-Length Tau or Its C-Terminal Fragments, Are Released from Neurons Independently of Cell Death

doi: 10.1523/JNEUROSCI.0387-15.2015

Figure Lengend Snippet: Free-floating tau is readily detected in CM from healthy iHCNs. A, Bright-field image of neurons 45 d after differentiation. Scale bar, 100 μm. Because the cells were almost confluent and it was difficult to distinguish one cell from another, an edge of lower density is shown. B, Western blot analysis of lysates with or without calf intestine alkaline phosphatase (CIP), 12.5 μg of total protein/lane, of 45 d postdifferentiation neurons (iHCNs) using anti-tau antibodies Tau12 (aa 6–18), K9JA (aa 243–441), Tau46 (aa 404–441), and the negative control antibody 46–4. Recombinant versions of all 6 human tau isoforms (Rec), 12 ng of total protein, and CIP-treated human brain extract (Hu), 4 μg of total protein, were loaded as controls. C, Medium collected from cultures after 48 h of conditioning was analyzed for tau, LDH, and β3T and the values presented are expressed relative to the amount of the corresponding protein detected in the lysates of the same cells. In 5 of 6 experiments, tau was released at significantly higher levels than LDH. No β3T was detected in CM. D, CM processed as described in Figure 1A. E, A crude exosomal pellet isolated from 35 ml of CM was further fractionated on a sucrose gradient according to Figure 1B. Tau was found in fractions with sucrose density of 1.10–1.16 g/ml (fractions 3–6).

Article Snippet: All tissue culture reagents were obtained from Invitrogen and, where appropriate, the medium used to condition cells was centrifuged (100,000 × g for 18 h and 4°C) to remove exosomes ( Théry et al., 2001 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Antibody Source Concentration Alix 3A9 Cell Signaling Technology 1 μg/ml Flotillin-1 18/Flotillin-1 BD Biosciences 1 μg/ml HIV glycoprotein 120 46-4 ATCC 1 μg/ml PrP ICSM35 D-Gen 0.5 μg/ml β3-tubulin ab68193 Abcam 1:250 dilution β3-tubulin TUJ1 Covance 5 μg/ml Tau (aa 6-18) Tau12 (human) EMD Millipore 0.1 μg/ml a Tau (aa 7-13) HJ9.4 (rodent) Gift from David Holtzman 1 μg/ml a Tau (aa 194-198) BT2 (human + rodent) Thermo Scientific 1 μg/ml a , b Tau ( aa 210-241) Tau5 (human + rodent) Cell Signaling Technology 1 μg/ml c Tau (aa 243-441) K9JA (human + rodent) DAKO 1 μg/ml a , b , c Tau (aa 404-441) Tau46 (human + rodent) Cell Signaling Technology 1 μg/ml a Misfolded tau (residues 7-9 + 313-322) MC1 Gift from Peter Davies 5 μg/ml a Open in a separate window Concentrations used for Western blotting: a 5 μg/ml for IP, b 2.5 μg/ml for ELISA, c 5 μg/ml for ELISA.

Techniques: Western Blot, Negative Control, Recombinant, Isolation

Journal: The Journal of Neuroscience

Article Title: C-Terminally Truncated Forms of Tau, But Not Full-Length Tau or Its C-Terminal Fragments, Are Released from Neurons Independently of Cell Death

doi: 10.1523/JNEUROSCI.0387-15.2015

Figure Lengend Snippet: Conditioned medium from healthy primary rat hippocampal neurons contain readily detectable levels of tau. A, Bright-field image of DIV 21 neurons. Scale bar, 50 μm. Because the cells were almost confluent and it was difficult to distinguish one cell from another, an edge of lower density is shown. B, Lysates (50 μg) of PRNs were treated with or without calf intestine alkaline phosphatase (CIP) and used for Western blotting with the anti-tau antibodies HJ9.4 (aa 7–13), BT2 (aa 194–198), K9JA (aa 243–441), Tau46 (aa 404–441), and the negative control antibody 46–4. Recombinant rodent tau 431 (Rec), 10 ng, and CIP-treated rat brain extract (Rat), 5 μg, were loaded as controls. C, Medium collected from DIV 21 neurons after 24 h of conditioning was analyzed for the presence of tau, LDH, and β3T. Tau was detected at levels disproportionate to the release of LDH and, in 5 of 8 experiments, the levels were significantly higher than LDH (p < 0.02). NS indicates that the levels of tau and LDH are not significantly different. β3T was not detected in the CM from any experiment. D, CM processed as described in Figure 1A. E, Crude exosomal pellet isolated from 35 ml of CM was further fractionated on a sucrose gradient according to Figure 1B. Tau was found in fractions with sucrose density 1.11–1.16 g/ml, fractions 3–6, consistent with exosomes.

Article Snippet: All tissue culture reagents were obtained from Invitrogen and, where appropriate, the medium used to condition cells was centrifuged (100,000 × g for 18 h and 4°C) to remove exosomes ( Théry et al., 2001 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Antibody Source Concentration Alix 3A9 Cell Signaling Technology 1 μg/ml Flotillin-1 18/Flotillin-1 BD Biosciences 1 μg/ml HIV glycoprotein 120 46-4 ATCC 1 μg/ml PrP ICSM35 D-Gen 0.5 μg/ml β3-tubulin ab68193 Abcam 1:250 dilution β3-tubulin TUJ1 Covance 5 μg/ml Tau (aa 6-18) Tau12 (human) EMD Millipore 0.1 μg/ml a Tau (aa 7-13) HJ9.4 (rodent) Gift from David Holtzman 1 μg/ml a Tau (aa 194-198) BT2 (human + rodent) Thermo Scientific 1 μg/ml a , b Tau ( aa 210-241) Tau5 (human + rodent) Cell Signaling Technology 1 μg/ml c Tau (aa 243-441) K9JA (human + rodent) DAKO 1 μg/ml a , b , c Tau (aa 404-441) Tau46 (human + rodent) Cell Signaling Technology 1 μg/ml a Misfolded tau (residues 7-9 + 313-322) MC1 Gift from Peter Davies 5 μg/ml a Open in a separate window Concentrations used for Western blotting: a 5 μg/ml for IP, b 2.5 μg/ml for ELISA, c 5 μg/ml for ELISA.

Techniques: Western Blot, Negative Control, Recombinant, Isolation

Journal: The Journal of Neuroscience

Article Title: C-Terminally Truncated Forms of Tau, But Not Full-Length Tau or Its C-Terminal Fragments, Are Released from Neurons Independently of Cell Death

doi: 10.1523/JNEUROSCI.0387-15.2015

Figure Lengend Snippet: Aβ-induced cell compromise increases the amount of truncated tau in medium from primary rat hippocampal neurons. A, Aggregation of SEC-isolated Aβ(1–42) was followed using a continuous thioflavin T (ThT)-binding assay and fluorescence values are expressed in relative fluorescence units and plotted versus time. The ½tmax point at which sample was collected and used for toxicity experiments is indicated by the red arrow and the inset shows a negatively stained transmission EM of ½tmax Aβ(1–42). Scale bar, 100 nm. B, LDH activity in PRN CM after 1, 3, 5, and 7 d of treatment with 20 μm ½tmax Aβ(1–42); (■), 20 μm glutamate (▴), and vehicle (10.9 mm HEPES, pH 7.8; ●). Differences in LDH levels between the vehicle control and test conditions were compared on each day using the Student's t test. Glutamate caused an elevation in tau on all days (p < 0.01), whereas Aβ treatment increased tau only on days 5 and 7 (p < 0.01). Insets show MAP2-stained neurons after 7 d of treatment with 20 μm ½tmax Aβ(1–42) and 10.9 mm HEPES, pH 7.8. C, Tau detected by BT2-Tau5 (mid-region) ELISA in PRN CM after 1, 3, 5, and 7 d of treatment with 20 μm ½tmax Aβ(1–42); (■), 20 μm glutamate (▴), and vehicle (10.9 mm HEPES, pH 7.8; ●). Differences in tau concentration between the vehicle control and test conditions were compared on each day using the Student's t test. Glutamate treatment (▴) caused a significant elevation in tau on all days (p < 0.01), whereas Aβ(1–42) (■) caused a significant increase only on days 5 and 7 (p < 0.01). D, E, Tau detected by the CT1 (K9JA-K9JA; D) and CT2 (K9JA-Tau46; E) ELISAs evinced elevation on days 1, 3, 5, and 7 in medium from cells treated with glutamate (▴; p < 0.05) and on days 5 and 7 when cells were treated with Aβ(1–42) (■; p < 0.02). F, Tau detected in PRN CM by the FL (HJ9.4-Tau46) ELISA showed no difference between cultures treated with vehicle or glutamate on any day, whereas the levels of FL tau were elevated in CM from neurons treated with Aβ(1–42) (■) compared with vehicle (10.9 mm HEPES, pH 7.8; ●) on day 7 (p < 0.05). G, ADDLs were prepared as described in the Materials and Methods and characterized by aSEC (void volume indicated with an arrow) and negative contrast EM. Scale bar, 100 nm. H, Tau detected by BT2-Tau5 (mid-region) ELISA in PRN CM after 1, 3, 5, and 7 d of treatment with 0.5 μm ADDLs (♦), 20 μm glutamate (▴), and vehicle (10.9 mm HEPES, pH 7.8; ●). Differences in tau concentration between the vehicle control and test conditions were compared on each day using the Student's t test. Glutamate treatment (▴) caused a significant elevation in tau on all days (p < 0.05), whereas ADDLs (♦) did not cause a significant increase on any day (p > 0.05). I, Tau detected by the CT1 (K9JA-K9JA) ELISA in PRN CM after 1, 3, 5, and 7 d of treatment with 0.5 μm ADDLs (♦), 20 μm glutamate (▴), and vehicle (10.9 mm HEPES, pH 7.8; ●). Glutamate treatment (▴) caused a significant elevation in tau on all days (p < 0.01), whereas ADDLs (♦) did not cause a significant increase on any day (p > 0.05). The data shown are derived from a minimum of six wells per treatment.

Article Snippet: All tissue culture reagents were obtained from Invitrogen and, where appropriate, the medium used to condition cells was centrifuged (100,000 × g for 18 h and 4°C) to remove exosomes ( Théry et al., 2001 ). table ft1 table-wrap mode="anchored" t5 caption a7 Antigen Antibody Source Concentration Alix 3A9 Cell Signaling Technology 1 μg/ml Flotillin-1 18/Flotillin-1 BD Biosciences 1 μg/ml HIV glycoprotein 120 46-4 ATCC 1 μg/ml PrP ICSM35 D-Gen 0.5 μg/ml β3-tubulin ab68193 Abcam 1:250 dilution β3-tubulin TUJ1 Covance 5 μg/ml Tau (aa 6-18) Tau12 (human) EMD Millipore 0.1 μg/ml a Tau (aa 7-13) HJ9.4 (rodent) Gift from David Holtzman 1 μg/ml a Tau (aa 194-198) BT2 (human + rodent) Thermo Scientific 1 μg/ml a , b Tau ( aa 210-241) Tau5 (human + rodent) Cell Signaling Technology 1 μg/ml c Tau (aa 243-441) K9JA (human + rodent) DAKO 1 μg/ml a , b , c Tau (aa 404-441) Tau46 (human + rodent) Cell Signaling Technology 1 μg/ml a Misfolded tau (residues 7-9 + 313-322) MC1 Gift from Peter Davies 5 μg/ml a Open in a separate window Concentrations used for Western blotting: a 5 μg/ml for IP, b 2.5 μg/ml for ELISA, c 5 μg/ml for ELISA.

Techniques: Isolation, Binding Assay, Fluorescence, Staining, Transmission Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Derivative Assay

Journal: Alzheimer's & Dementia : Translational Research & Clinical Interventions

Article Title: Novel antibody against low‐n oligomers of tau protein promotes clearance of tau in cells via lysosomes

doi: 10.1002/trc2.12097

Figure Lengend Snippet: Analysis of the specificity of anti‐tau oligomer antibodies. Anti‐tau oligomer monoclonal antibodies exhibit differential abilities to bind to different tau species or constructs. A, Representative dot blot images show the affinity of antibodies to tau monomers, oligomers, and fibrils. 50 ng/well of protein was loaded on the polyvinylidene difluoride membrane, and 3 µg of antibody was used to detect the protein. Row 1a–j: A representative blot (pan‐tau polyclonal antibody K9JA) acts as a positive control that detects all tau constructs and assembly forms. By contrast, different monoclonal antibodies have different specificities and affinities to different constructs and assembly forms of tau. Row 2: Antibody 2B10 shows high specificity for the oligomer‐enriched sample (containing 1% monomers, 99% low‐n oligomers; blot 2c, green box) whereas antibody 6H1 (row 3) show high affinity to crude oligomers (containing 35% monomers, 63% low‐n oligomers, 2% high‐n oligomers; blot 3b) and aggregates (blot 3d, pink box) of the repeat domain (RD) of tau with the ΔK280 mutation. B, Immunocytochemistry of monoclonal antibodies detects tau with high specificity in cell models. 2B10 (1:100) and 6H1 (1:100) antibodies (green) detected tau in N2a cells expressing Tau RDΔK (image 4, 7) but with minimal or no staining in N2a‐wt cells (image 1; no signal for either 2B10 [shown] or 6H1 [not shown]; anti‐rat CF488). Pan tau K9JA antibody (1:1000; anti‐rabbit Alexa 555) was used as a total tau antibody (images 2, 5, 6) co‐localizing with anti‐tau oligomer antibodies (image 6, 9). C, Fluorescence imaging of free‐floating mouse brain sections probed with 2B10 antibody (anti‐rat CF488) showing tau staining mostly in the soma and some dendritic processes of CA1 and CA3 neurons in the transgenic mouse expressing Tau RDΔK (image 3, 4) versus non‐transgenic controls (image 1, 2). 2B10 antibody (1:100) does not show any staining in the control brain (image 1, 2) owing to its high specificity to Tau RDΔK . Abbreviations: Mono, monomer; Oligo, cross‐linked and purified oligomers; Oligo*, not cross‐linked and crude oligomers; Agg, Aggregates; RD, repeat domain; FL, full length

Article Snippet: Commercial antibodies used in the current study include K9JA (DAKO #A0024), anti‐rabbit AMCA (Dianova #711‐155‐152), anti‐rat CF488 (VWR #20027), anti‐rabbit Alexa 555 (abcam #ab150066).

Techniques: Construct, Dot Blot, Positive Control, Mutagenesis, Immunocytochemistry, Expressing, Staining, Fluorescence, Imaging, Transgenic Assay, Purification

Journal: Alzheimer's & Dementia : Translational Research & Clinical Interventions

Article Title: Novel antibody against low‐n oligomers of tau protein promotes clearance of tau in cells via lysosomes

doi: 10.1002/trc2.12097

Figure Lengend Snippet: Antibodies promote tau entry to lysosomes for clearance. 60 µg/mL of Alexa647 labeled 2B10 (red) or immunoglobulin G (IgG) control (red) antibodies applied extracellularly for 24 hours with 75 nM of Lysotracker dye (green) on N2a‐Tau RDΔK cells induced with doxycycline. A, Cells fixed and probed with antibody K9JA (blue) (anti‐rabbit AMCA) to detect Tau RDΔK (image 3, 7). Lysotracker dye stained lysosomes (images 1, 5). 2B10 and IgG control antibodies were taken up by cells and compartmentalized into puncta (images 2, 6). 2B10 antibody shows co‐localization with tau and lysosomes (image 4, white arrows), whereas the IgG control antibody shows co‐localization only with lysosomes but not with tau (image 8). B, Quantification of the triple co‐localized (tau+antibody in lysosomes) puncta per cell shows increased localization of tau and 2B10 in lysosomes. n = 3; Unpaired t test; **** P = < .0001

Article Snippet: Commercial antibodies used in the current study include K9JA (DAKO #A0024), anti‐rabbit AMCA (Dianova #711‐155‐152), anti‐rat CF488 (VWR #20027), anti‐rabbit Alexa 555 (abcam #ab150066).

Techniques: Labeling, Staining