Immunohistochemistry analyses were performed on whole mount fly larvae, cultured neurons and on mouse tissue sections collected from OCT-embedded tissues. Primary antibodies used were anti-Tuj1 (Covance, MMS-435P, 1:1,000), anti-Tau (Millipore, MAB3420, 1:1,000), anti-RTCA (Sigma, SAB2102059 and HPA027982, 1:200), anti-CTIP2 (Abcam, ab18465, 1:2,000), anti-NF200 (Sigma, N4142, 1:200), anti-ED1 (Abcam, ab31630, 1:200), anti-β-Galactosidase (MP Biomedicals cappel, 55976, 1:5,000) and anti-NeuN (Millipore, MAB377, 1:200). A custom-made antibody against the entire dRtca protein was generated (Thermo, 1:200). LacZ staining was performed using the Senescence β-Galactosidase Staining Kit (Cell signaling) according to manufacturer’s instructions.
Mab3420
Manufactured by Merck Group
Sourced in United States
MAB3420 is a laboratory equipment product manufactured by Merck Group. It is designed for specific laboratory applications, but a detailed description cannot be provided while maintaining an unbiased and factual approach. Additional information about the core function and intended use of this product is not available.
Automatically generated - may contain errors
Lab products found in correlation
Mab377, by Merck Group (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
T2200, by Merck Group (3 mentions)
Ab5622, by Merck Group (3 mentions)
Mms 435p, by Merck Group (2 mentions)
Ab31630, by MP Biomedicals (2 mentions)
Sab2102059, by Abcam (2 mentions)
Hpa027982, by Abcam (2 mentions)
Ab18465, by Merck Group (2 mentions)
Senescence β galactosidase staining kit, by Cell Signaling Technology (2 mentions)
N4142, by Abcam (2 mentions)
Ab15452, by Merck Group (2 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (2 mentions)
Ab6556, by Abcam (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
A11001, by Thermo Fisher Scientific (2 mentions)
Abn454, by Merck Group (2 mentions)
Triton x, by Merck Group (2 mentions)
Anti homer1, by Synaptic Systems (2 mentions)
38 protocols using mab3420
1
Immunohistochemistry Analysis of Neuronal Markers
2
Analyzing Ilf3 Mutant Neuron Development
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Dissociated AMY neurons were prepared from Ilf3+/+ and Ilf3ΔPrLD/ΔPrLD littermates at embryonic day 16-18 (E16-18). Neurons were plated at a density of 5.2 × 103 cells/cm2 onto poly-D-lysine-coated coverslips (Matsunami, Osaka, Japan) in Neurobasal-A medium (Thermo Fisher Scientific) containing B-27 supplement (Thermo Fisher Scientific), 0.5 mM glutamine, and 25% Neuron culture medium (FUJIFILM Wako Pure Chemical). Cultured neurons were incubated at 37°C in a 5% CO2 incubator. They were fixed with 3.7% formaldehyde in PBS for 10 min at 3, 5, and 7 days in vitro (DIV).
Immunostaining was performed as described above. The primary antibodies used were anti-MAP2 rabbit polyclonal antibody (ab32454, Abcam) and anti-Tau-1 mouse monoclonal antibody (MAB3420, Sigma-Aldrich). Secondary antibodies were Alexa488-conjugated anti-rabbit IgG antibody (Thermo Fisher Scientific) and Cy3-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch, West Grove, PA). Fluorescence images were acquired using the A1 confocal laser scanning microscope with a 20× or 40× objective. Serial images acquired at 0.5 μm steps were z-stacked. Concentric circles were drawn at 30 μm intervals around the nucleus for Sholl analysis, where the number of intersections was counted. One of the neurites with the strongest tau staining was determined to be the axon.
Immunostaining was performed as described above. The primary antibodies used were anti-MAP2 rabbit polyclonal antibody (ab32454, Abcam) and anti-Tau-1 mouse monoclonal antibody (MAB3420, Sigma-Aldrich). Secondary antibodies were Alexa488-conjugated anti-rabbit IgG antibody (Thermo Fisher Scientific) and Cy3-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch, West Grove, PA). Fluorescence images were acquired using the A1 confocal laser scanning microscope with a 20× or 40× objective. Serial images acquired at 0.5 μm steps were z-stacked. Concentric circles were drawn at 30 μm intervals around the nucleus for Sholl analysis, where the number of intersections was counted. One of the neurites with the strongest tau staining was determined to be the axon.
3
Immunofluorescence Staining of Neuronal Markers
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The following antibodies were used: mouse Tau-1 (Chemicon, MAB3420, 1:200), mouse anti-MAP2 (SigmaAldrich, M4403, 1:1500), rabbit anti-MAP2 (Abcam, ab32454, 1:1000), mouse anti-Ankyrin-G (Antibodies Inc., 75–146, 1:100), rabbit anti-Plexin-A1 (Abcam, ab23391, 1:1000), mouse anti-GFP (Covance, MMS-118P, 1:1000), mouse anti-FLAG M2 (SigmaAldrich, F3165, 1:1000), anti-Rap1 (Upstate, #07–916, 1:200), anti-Sema3A (Abcam, ab23393, 1:200), SMI-312 (BioLegend, 837904, 1:200) and goat secondary antibodies labeled with Alexa-350 (Molecular Probes, 1:200), −488 (1:800) or −594 (1:800). Nuclei were stained with Hoechst 33342 (Molecular Probes, 1:6000).
4
Immunofluorescence Staining of Transfected Cells
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Transfected cells or neurons were grown on glass cover slips coated with poly L-Lysine. Twenty-four hours after transfection for MN1 and on DIV2 for primary neurons, cells were fixed with 4% PFA for 2 min followed by ice cold methanol for 3 min and three washes with 1× PBS. Blocking was done using 5% goat serum in 1× PBS. The following primary antibodies were used: rabbit polyclonal CAMTA1 (Sigma-HPA036343) (1:100), rabbit polyclonal MID1IP1 (Sigma- HPA038816) (1:100), mouse monoclonal V5 (Invitrogen- 46–1157) (1:100), mouse monoclonal Tau1 (Millipore- MAB3420) (1:100) and mouse monoclonal Acetylated-Tubulin (Sigma- T7451) (1:750). Coverslips were incubated with primary antibodies in blocking solution at 4°C overnight. Coverslips were then washed three times with 1× PBS and incubated with secondary antibodies in blocking solution at room temperature for 2 h in dark. Coverslips were then washed three times with 1× PBS, submerged in MilliQ water and mounted on glass microscope slides with Fluoromount-G (Southern Biotech). Cells were imaged using an Olympus Fluoview 1000 microscope with 60× objective, using similar acquisition settings for laser power, offset and detector gain across conditions.
5
Immunofluorescence and Immunoblotting Protocols
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For immunofluorescence studies, the primary antibodies used were mouse anti-FLAG (M2; 1:1000; Sigma F1804); chicken anti-MAP2 (1:1000; Millipore AB15452); mouse anti-tau (1:1000; Millipore MAB3420) and rabbit anti-β-tubulin-III (1:400; Sigma T2200). The secondary antibodies used for immunofluorescence were all sourced from Thermo Fisher (Waltham, MA, USA) and include; donkey anti-mouse IgG Alexa555 (1:2000 A31570); donkey anti-mouse IgG Alexa647 (1:1000 A31571) and donkey anti-rabbit IgG Alexa555 (1:2000 A31572). For immunoblotting, the primary antibodies were rabbit anti-hIQSEC2 (1:2000; details in Supplementary Methods ), mouse β-actin (AC-74; 1:20 000; Sigma A2228) and rabbit anti-IQSEC3 (1:500; Abcam AB107853). Secondary antibodies from DAKO (Santa Clara, CA, USA) were goat anti-mouse HRP (1:2000 P0447) and goat anti-rabbit HRP (1:2000 P0448).
6
Immunohistochemistry Analysis of Neuronal Markers
7
Immunostaining Protocol for Neuronal and Fibroblast Cells
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For primary hippocampal neurons and NIH/3T3 cells, cells were rinsed with PBS and fixed with 4% PFA for 10 minutes. Cell were permeabilized with PBST (PBS with 0.2% Triton X-100) for 10 minutes and blocked with 1% BSA in PBST for 30 minutes. Primary antibodies were incubated at 4℃ for overnight. Antibodies against Tau1 (Millipore, MAB3420, monoclonal), FLAG (Sigma, F7425, polyclonal), and MTCO1 (Abcam, ab203912, monoclonal) were used for immunostaining. The cells were washed three times with PBS and incubated with Alexa Fluor secondary antibodies (Invitrogen).
Then these cells were washed three times with PBS and coverslips were mounted on slide glasses.
Images were taken by using LSM780 confocal microscopy. For Neuro2A, cells were washed with icecold PBS followed by fixation using 4% PFA/sucrose for 15 minutes. After 3 times washing with PBS, cells were permeabilized with PBST (PBS with 0.5% Triton X-100) for 15 minutes and blocked with 1% BSA in PBS for 1 hour. Primary antibodies were incubated at 4℃ for overnight. Cells were washed 3 times with PBS and incubated with Alexa Fluor secondary antibodies (Invitrogen) for 1 hour at room temperature. Cells were washed three times with PBS and coverslips were mounted on slide glasses.
Antibodies against GFP (Abcam, ab 6556, polyclonal) and MT-CO1 (Abcam, ab14705, monoclonal) were used for immunostaining.
Then these cells were washed three times with PBS and coverslips were mounted on slide glasses.
Images were taken by using LSM780 confocal microscopy. For Neuro2A, cells were washed with icecold PBS followed by fixation using 4% PFA/sucrose for 15 minutes. After 3 times washing with PBS, cells were permeabilized with PBST (PBS with 0.5% Triton X-100) for 15 minutes and blocked with 1% BSA in PBS for 1 hour. Primary antibodies were incubated at 4℃ for overnight. Cells were washed 3 times with PBS and incubated with Alexa Fluor secondary antibodies (Invitrogen) for 1 hour at room temperature. Cells were washed three times with PBS and coverslips were mounted on slide glasses.
Antibodies against GFP (Abcam, ab 6556, polyclonal) and MT-CO1 (Abcam, ab14705, monoclonal) were used for immunostaining.
8
Immunostaining Assay for Neurite Outgrowth
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To explore the neurite outgrowth, the immunostaining was performed as follows. Cultured neurons were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 20 min at 4 °C and wash three times with PBS for 5 min each. To permeate the cell membrane, 1% Triton X-100 (Sigma-Aldrich) was used for 5 min at room temperature, followed by PBS washing. For blocking nonspecific binding, the sample was treated with 6% bovine serum albumin (BSA; Sigma-Aldrich) for 30 min. After washing with PBS, it was reacted with primary antibodies diluted in 1.5% BSA solution for 2 h at 37 °C. The following primary antibodies were used: anti-MAP2 (1:500, M3696, Sigma-Aldrich), anti-tau-1 (1:500, MAB3420, Merck Millipore, MA, USA), anti-beta-III-tubulin (1:500, T2200, Sigma-Aldrich). Secondary antibodies (Alexa Fluor 488 and 594, 1:500, A11001, A11008, A11012 and A11032, Invitrogen; Thermo Fisher Scientific) diluted in 1.5% BSA were loaded for 30 min at 37 °C. To characterize damage of a biomolecule on the NIR-illuminated surface, anti-laminin (1:500, L9393, Sigma-Aldrich) was used with the same procedure except that there is no cell fixation and permeabilization step. An inverted microscope (IX71; Olympus) with a digital camera (DP71; Olympus) was used to take fluorescence and phase-contrast images.
9
Antibody Validation for Western Blot
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The primary antibodies used for WB were PTPN13 (1:1000, LS-C148268, LifeSpan BioSciences), calpain-1 (1:800, 2556, CST), calpain-2 (1:1000, LS-B12657, LifeSpan BioSciences), tau-5 (1:2000, AHB0042, Thermo Fisher Scientific), tau-1 (1:2000, MAB3420, EMD Millipore), anti-Tau oligomer T22 (1:300, ABN454, EMD Millipore), phospho-tau Ser202/Thr205 AT8 (1:1000, MN1020, Thermo Fisher), phospho-Tau Thr231 AT180 (1:1000, MN1040, Thermo Fisher), phospho-tau Ser416 (1:1000, p1573–416, PhosphoSolutions), c-Abl (1:1000, 2862, CST), spectrin (1:1000, MAB1622, EMD Millipore), phosphotyrosine (05–321, EMD Millipore), Phospho-c-Abl Tyr245 (1:800, 2861, CST), Phospho-c-Abl Tyr412 (1:800, 2865, CST) and actin (1:10000, A2228, Sigma-Aldrich). The secondary antibodies used for WB were IRDye 680RD goat anti-rabbit (1:10000, 926–68071, LI-COR) and IRDye 800CW goat anti-mouse (1:10000, 926–32210, LI-COR).
10
Immunofluorescence Staining of Neurons
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Immunofluorescence assays were performed as previously described.27 (link) Briefly, neurons were fixed in 4% paraformaldehyde supplemented with 4% sucrose for 40 min at 4°C and blocked with blocking buffer (3% BSA in TBS). Sections were then incubated with rabbit anti-GFP (1:1000; cat no. Ab290, Abcam) and mouse anti-tau-1 antibody (1:500; cat no. MAB3420, EMD Millipore) overnight at 4°C. After washing three times with TBST (0.1% Triton X-100 in TBS), sections were labelled with appropriate fluorescent-tagged secondary antibodies (goat anti-mouse/rabbit IgG FITC, 1:1000; cat no. ab150115, Abcam) for 1 h at room temperature, and then neurons were mounted on glass slides using Fluoro Gel II containing DAPI for confocal microscopy studies (LSM 700; Zeiss GmbH, Germany).
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