Immunohistochemistry was performed as described previously (Costa Mdo et al., 2013 (link); Zeng et al., 2013 (link)). Briefly, free floating sections were labeled with goat anti-N-terminal HTT (1:250; Santa Cruz Biotechnology, Santa Cruz, CA) or mouse anti-ATXN3 (1H9) (1:1000, MAB5360; Millipore). Immunostaining was performed using the Vectastain Elite Kit (Vector Laboratories, Burlingame, CA) or Vector MOM immunodetection kit (Vector Laboratories) when a mouse primary antibody was used. Sections were developed in ImmPACT DAB (Vector Laboratories), mounted on Superfrost slides (Fisher Scientific, Pittsburgh, PA) and air dried after dehydration with graded ethanol and xylene. Coverslips were affixed with DPX (Electron Microscopy Sciences, Hatfield PA, USA). All sections were imaged with an Olympus BX51 microscope (Olympus, Center Valley, PA).
Mab5360
Manufactured by Merck Group
Sourced in United States
MAB5360 is a laboratory instrument used for the detection and quantification of specific biomolecules. It employs monoclonal antibody technology to perform sensitive and selective assays. The core function of MAB5360 is to enable researchers to accurately measure the presence and levels of target analytes in samples.
Automatically generated - may contain errors
Lab products found in correlation
Mab1574, by Merck Group (3 mentions)
Vector m o m immunodetection kit, by Vector Laboratories (2 mentions)
Goat anti n terminal htt, by Santa Cruz Biotechnology (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Dibutylphthalate polystyrene xylene (dpx), by Electron Microscopy Sciences (1 mentions)
Vectastain elite kit, by Vector Laboratories (1 mentions)
Superfrost slide, by Thermo Fisher Scientific (1 mentions)
Immpact dab, by Vector Laboratories (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Rabbit anti ubqln2, by Novus Biologicals (1 mentions)
Guinea pig anti p62 c terminal, by Progen Biotechnik (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Lysosensor green, by Thermo Fisher Scientific (1 mentions)
Sc 362262, by Santa Cruz Biotechnology (1 mentions)
Donkey anti goat secondary antibody, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Envision multilabel reader, by PerkinElmer (1 mentions)
Dab substrate, by Vector Laboratories (1 mentions)
Prolong gold medium, by Thermo Fisher Scientific (1 mentions)
A1 high sensitivity confocal microscope, by Nikon (1 mentions)
16 protocols using mab5360
1
Immunohistochemical Labeling of Protein Targets
2
Immunohistochemical Analysis of Neurodegenerative Proteins
3
Fluorescent Labeling of Cellular Organelles and Protein Markers
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Live cell imaging of lysosome intensity was done using LysoSensor Green (Life Technologies L7535). Dye was mixed in culture media at a 1:1000 dilution prior to adding media to wells. Cells were then incubated for 5 min in LysoSensor solution. Media was then aspirated and cells were washed 2× with PBS. All imaging was done within one hour of staining.
To assay for pluripotency markers, hPSC cultures were fixed using 4% PFA and incubated at room temperature for 10 min. Cells were then permeabilized using 0.05% Triton X-100 and incubated for 10 min. Following two washes with 5% goat serum, NANOG antibody (R&D Systems AF1997, 1:200) and TRA-1-60 antibody (Millipore MAB5360, 1:150), was added to cells and incubated overnight at 4 °C. The next day, cells were rinsed twice with 5% goat serum and then incubated with a donkey anti-goat secondary antibody (Life Technologies A11055 1:500) for 1 h at room temperature. Cells were then washed twice with PBS and mounted for imaging.
Cardiomyocyte cultures were processed in the same manner as above. After permeabilization cells were incubated with anti-sarcomeric alpha-actinin (Abcam ab68167 1:250) overnight at 4 °C. The next day, cells were rinsed twice with 5% goat serum and then incubated with a goat anti-rabbit secondary antibody (Santa Cruz Biotech sc-362262, 1:500)
To assay for pluripotency markers, hPSC cultures were fixed using 4% PFA and incubated at room temperature for 10 min. Cells were then permeabilized using 0.05% Triton X-100 and incubated for 10 min. Following two washes with 5% goat serum, NANOG antibody (R&D Systems AF1997, 1:200) and TRA-1-60 antibody (Millipore MAB5360, 1:150), was added to cells and incubated overnight at 4 °C. The next day, cells were rinsed twice with 5% goat serum and then incubated with a donkey anti-goat secondary antibody (Life Technologies A11055 1:500) for 1 h at room temperature. Cells were then washed twice with PBS and mounted for imaging.
Cardiomyocyte cultures were processed in the same manner as above. After permeabilization cells were incubated with anti-sarcomeric alpha-actinin (Abcam ab68167 1:250) overnight at 4 °C. The next day, cells were rinsed twice with 5% goat serum and then incubated with a goat anti-rabbit secondary antibody (Santa Cruz Biotech sc-362262, 1:500)
4
Quantification of Mutant Ataxin-3 Levels
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The TR-FRET assay was performed as described before.41 (link) Brain tissues were lysed by sonication in 10× v/w of ice-cold lysis buffer (PBS + 1% Triton X-100 + 1× protease inhibitor cocktail [Roche]). Anti-ataxin-3 clone 1H9 (MAB5360, Millipore) was labeled with donor Lumi4-Tb-fluorophore (Cisbio). Anti-ployglutamine expansion marker clone 5TF1-1C2 (MAB1574, Millipore) was labeled with D2 acceptor fluorophore (Cisbio). The combination of 1H9 and 5TF1-1C allow detection of more than 37 glutamine repeats. After optimization of antibody titers and incubation conditions, quantification of mutant ataxin-3 levels was performed in low-volume polystyrene 384 microtiter plates (Greiner Bio-One) with 5 μL of sample volume and addition of 1 μL of antibody solution (50 mM NaHPO4 + 400 mM NaF + 0.1% BSA + 0.05% Tween 20 + 1 ng/mL 1H9-Tb + 10 ng/mL 1C2-D2). Plates were then incubated at 4°C for 20 h and analyzed by time-resolved fluorescence at 620 nm and 665 nm on an EnVision multilabel reader (PerkinElmer).
5
Quantification of ATXN3 Aggregation in Mouse Brain
6
Immunostaining of SCA3 Neural Progenitor Cells
7
Multimodal Immunofluorescence Imaging
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After rewarming, the sections were incubated in 0.3% Triton™ X-100 and Immunol Staining Blocking buffer (Beyotime, China) for 1 h at room temperature. The sections were then incubated overnight at 4 °C with the following primary antibodies: mouse anti-calbindin D28K (1:500, catalog number C9848, Sigma-Aldrich, USA), mouse anti-ATXN3 (1H9) (1:200, catalog number MAB5360, Millipore, USA), rabbit anti-glial fibrillary acidic protein (GFAP) (1:200, catalog number ab48050, Abcam, USA), and rat anti-myelin basic protein (MBP) (1:200, catalog number ab7349, Abcam, USA), followed by incubation with the following secondary antibodies: goat-anti mouse IgG (H+L), F(ab’)2 fragment (Alexa Fluor® 488 conjugated) antibody (1:300, catalog number 4408S, Cell Signaling, USA), goat-anti mouse IgG (H+L), F(ab’)2 fragment (Alexa Fluor® 555 conjugated) antibody (1:300, catalog number 4409S, Cell Signaling), goat-anti rabbit IgG (H+L), F(ab’)2 fragment (Alexa Fluor® 488 conjugated) antibody (1:300, catalog number 4412S, Cell Signaling), and goat-anti rat IgG (H+L), F(ab’)2 fragment (Alexa Fluor® 488 conjugated) antibody (1:300, catalog number 4416S, Cell Signaling) for 1 h at room temperature. Finally, DAPI was used to identify the nuclei. Immunofluorescence images were obtained with a confocal laser scanning microscope (Leica) or a two-photon confocal laser scanning microscope (Leica).
8
Western Blot Analysis of Protein Markers
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A total of 40 µg of protein was mixed with 0.1 M DTT (D1532, Invitrogen) and 4× LDS sample buffer (NP0007, Invitrogen) and heated at 95 °C for 5 min to prepare denatured-reducing protein. The denatured-reducing protein was applied to electrophoresis on a polyacrylamide gel and then transferred to polyvinyldene fluoride membranes. For blocking the nonspecific binding, 5% skim milk was used, and followed by the primary antibodies, incubating at 4 °C overnight. At 5 min of 1× PBST, washing steps were then performed for 3 times on membranes, and followed by 1 h incubation of HRP-conjugated secondary antibody at room temperature. After the final 3 times of 1× PBST washing steps, the chemiluminescence detection was performed, and the intensity of the signal was detected by an iBright CL1000 Imaging System (Thermo Scientific).
The primary antibodies used in this study were as follows: ATXN3 (1:1000; MAB5360, Millipore), calpain 1 (1:5000; GTX102340, GeneTex, Irvine, CA, USA), calpain 2 (1:5000; GTX111809, GeneTex), calpastatin (1:5000; GTX645540, GeneTex), PARP1 (1:1000, MAB600, R&D), GAPDH (1:10,000, ab8245, Abcam), β-actin (1:5000; MAB8929, R&D), and HDAC2 (1:5000; GTX109642, GeneTex).
The primary antibodies used in this study were as follows: ATXN3 (1:1000; MAB5360, Millipore), calpain 1 (1:5000; GTX102340, GeneTex, Irvine, CA, USA), calpain 2 (1:5000; GTX111809, GeneTex), calpastatin (1:5000; GTX645540, GeneTex), PARP1 (1:1000, MAB600, R&D), GAPDH (1:10,000, ab8245, Abcam), β-actin (1:5000; MAB8929, R&D), and HDAC2 (1:5000; GTX109642, GeneTex).
9
Immunohistochemical Analysis of Ataxin-3 and Associated Markers
10
Western Blot Analysis of Ataxin-3 Proteins
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Anti-ataxin-3 (mouse monoclonal 1H9, MAB5360, 1:500–1000; Millipore), anti-MJD (rabbit polyclonal, 1:15,000 Paulson et al., 1997 (link)), anti-HA (rabbit monoclonal C29F4, 1:500–1000; Cell Signaling Technology), anti-V5 (mouse monoclonal R960-25, 1:500–1000; ThermoFisher), anti-tubulin (mouse monoclonal T5168, 1:10,000; Sigma-Aldrich), anti-lamin (mouse monoclonal ADL84.12–5, 1:1000; Developmental Studies Hybridoma Bank), anti-HSPA8/Hsc70-4 (rabbit monoclonal D12F2, 1:1000; Cell Signaling Technology), peroxidase conjugated secondary antibodies (goat anti-mouse, goat anti-rabbit, 1:5000–10,000; Jackson Immunoresearch).
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