Cells were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature (RT). For permeabilization and non-specific antibody blocking, 0.3% Triton-X containing 5% bovine serum albumin (BSA) (Sigma) in PBS was added for 60 min. Primary antibodies were made up in 5% BSA and then applied overnight at 4°C. Primary antibodies used were SMI-32 (BioLegend; 801701; mouse; 1:1000), ChAT (Millipore; AB144P; goat; 1:100), βIII-tubulin (abcam; ab41489; chicken; 1:1000), TDP-43 (ProteinTech; 12892-1-AP; rabbit; 1:400), SFPQ (abcam; ab11825; mouse; 1:400), FUS (Santa Cruz; sc-47711; mouse; 1:200), hnRNPA1 (Cell Signaling; 8443S; rabbit; 1:500) and hnRNPK (Santa Cruz; sc-28380; mouse; 1:500). A species-specific Alexa Fluor-conjugated secondary antibody (Life Technologies) at 1:1000 dilution in 5% BSA was added for 90 min at RT in the dark. Cells were washed once in PBS containing DAPI, 4′,6-diamidino-2-phenylindole nuclear stain (1:1000) for 10 min.
Ab144p
Manufactured by Abcam
AB144P is an antibody affinity purified from rabbit serum. It is designed for use in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
A21202, by Thermo Fisher Scientific (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Hnrnpa1, by Cell Signaling Technology (1 mentions)
βiii tubulin, by Abcam (1 mentions)
Smi 32, by BioLegend (1 mentions)
Tdp 43, by Proteintech (1 mentions)
Hnrnpk, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ab18976, by Abcam (1 mentions)
Mab1195, by R&D Systems (1 mentions)
A11034, by Thermo Fisher Scientific (1 mentions)
Nbp128667, by Novus Biologicals (1 mentions)
A11003, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Dm5000b, by Leica (1 mentions)
Ab22450, by Abcam (1 mentions)
Ab36367, by Abcam (1 mentions)
Avidin biotin peroxidase detection system, by Vector Laboratories (1 mentions)
Prb 435p, by Merck Group (1 mentions)
10 protocols using ab144p
1
Immunofluorescence Assay for Neuronal Markers
2
Immunocytochemical Analysis of Stem Cell Markers
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Cells were fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.25% Triton X-100 and blocked with 5% FCS in PBS for 1 h. The fixed cells were incubated overnight at 4 °C in PBS + 1% FCS with antibodies mouse anti-OCT4 (1:500, ab18976, Abcam, Cambridge, MA), rabbit anti-SOX2 (1:500, MA516399, ThermoFisher), rabbit anti-TUJ1 (1:500, MAB1195, R&D system, Minneapolis, MN), rabbit anti-OLIG2 (1:500, NBP128667, Novus), rabbit anti-HB9 (1:500, ABN174, Millipore-Sigma, Temecula, CA), rabbit anti-CHAT (1:500, AB144P, Abcam, Cambridge, MA), and rabbit anti-cleaved caspase-3 (CC3, 1:500, 9669S, Cell Signaling Technology) followed by incubation with secondary antibodies: FITC-conjugated anti-mouse IgG (1:1000, A21202, Life Technologies), FITC-conjugated anti-rabbit IgG (1:1000, A11034, Life Technologies), Cy3-conjugated anti-mouse IgG (1:1000, A11003, Life Technologies), and Cy3-conjugated anti-rabbit IgG (1:1000, A11035, Life Technologies). The treated cells were covered with Slowfade antifade with DAPI (Life Technologies) for nuclear staining and covered with a glass coverslip. Images were captured with a fluorescence microscope (DM5000B, Leica, Wetzlar, Germmany).
3
Comprehensive Western Blot and Immunofluorescence Antibodies
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The following antibodies were used for western blot: anti-MHC3 (embryonic myosin) (1:500; F1.652, DHSB), anti-MHC8 (postnatal myosin) (1:500; DSHB, N3.36), anti-islet 1 (ISL1) (1:1,000; R&D, AF1837-SP), anti-doublecortin (DCX) (1:2,000; Cell Signaling Technology, 4604), anti-choline acetyltransferase (ChAT) (1:1,000, Merck, AB144P), anti-OCT4 (1:250; Stemgent, 09-0023), anti-GAPDH (1:5,000; Abcam, ab9485), anti-β-actin (1:5,000; Sigma, A5441), and anti-HTT (1:5,000; Sigma-Aldrich, MAB5492).
The following reagents were used for immunofluorescence assays: Hoechst 33342, BgTx (5 μg/ml; Thermo Fisher, B1196), anti-bassoon antibody (1:2,000; Synaptic Systems, 141 013), tetanus neurotoxin (TeNT) (2 μg/ml; Sigma, T3194-25UG), anti-neurofilament M antibody (1:1,000; Synaptic Systems, 171 204), anti-myosin heavy chain 1 (MHC 1) antibody (1:1,000; Millipore, 05-716), anti-mCherry antibody (1:5,000, Abcam, ab205402), anti-TOMM20 antibody (1:500; Abcam, ab186735), anti-HTT antibody (1:500; Merck, MAB5374, clone mEM48), anti-ChAT antibody (1:100; Merck, AB144P), anti-MAP2 antibody (1:2,000; Abcam, ab5392), anti-V5 antibody (1:800; Cell Signaling Technology, D3H8Q), and anti-NF2H3 antibody (1:40; DSHB, AB2314897). All the secondary antibodies were Alexa-conjugated and were obtained from Jackson ImmunoResearch. They were all used at 1:1,000 dilutions for 1 h at RT.
The following reagents were used for immunofluorescence assays: Hoechst 33342, BgTx (5 μg/ml; Thermo Fisher, B1196), anti-bassoon antibody (1:2,000; Synaptic Systems, 141 013), tetanus neurotoxin (TeNT) (2 μg/ml; Sigma, T3194-25UG), anti-neurofilament M antibody (1:1,000; Synaptic Systems, 171 204), anti-myosin heavy chain 1 (MHC 1) antibody (1:1,000; Millipore, 05-716), anti-mCherry antibody (1:5,000, Abcam, ab205402), anti-TOMM20 antibody (1:500; Abcam, ab186735), anti-HTT antibody (1:500; Merck, MAB5374, clone mEM48), anti-ChAT antibody (1:100; Merck, AB144P), anti-MAP2 antibody (1:2,000; Abcam, ab5392), anti-V5 antibody (1:800; Cell Signaling Technology, D3H8Q), and anti-NF2H3 antibody (1:40; DSHB, AB2314897). All the secondary antibodies were Alexa-conjugated and were obtained from Jackson ImmunoResearch. They were all used at 1:1,000 dilutions for 1 h at RT.
4
Immunofluorescence and Immunochemistry Protocols
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Immunofluorescence and immunochemistry were performed as previously described (Cosset et al., 2015 (link), 2016 (link)). The following primary antibodies were used: mouse anti-neuronal nuclei-specific protein (NeuN) (Chemicon;MAB377), rabbit anti-βIII-tubulin (Covance;PRB435P), goat anti-ChaT (Chemicon; AB144P), rabbit anti-HB-9 (Abcam; ab922606), rabbit anti-ISLET1 (Abcam; ab22450), goat anti-GALR3 (Abcam), mouse anti-EV-71 (Abcam; ab36367), and mouse anti-PV-3 (Abcam; ab22450). Alexa Fluor (555 and 488)-labeled antibodies from goat or donkey against mouse, goat, or rabbit (Molecular Probes) were used as secondary antibodies. Cell nuclei were stained with DAPI (4, 6-diamidino-2-phenylindole). For IHC, biotin-conjugated anti-rabbit IgG or anti-goat IgG were used and developed using avidin-biotin peroxidase detection system (Vector Labs) with 3,3′-diaminobenzidine substrate (DAB, Sigma-Aldrich) after 2 min of incubation.
5
Immunostaining of Mouse Brain Sections
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Mice were anesthetized with 1% pentobarbital sodium and perfused transcardially with 0.1 mol/L PBS followed by 4% paraformaldehyde (PFA) in 0.1 mol/L PBS. The brain was removed and post-fixed overnight in 4% PFA at 4℃, then equilibrated in 30% sucrose. After embedding in OCT, the brain tissue was cut at 30 µm on a Leica cryostat or cooled-stage microtome and stored in 0.1 mol/L PBS at 4℃. For immunostaining, sections were incubated with 0.5% Triton X-100 (PBST) for 30 min at room temperature and 5% BSA in PBST for 30 min at room temperature, then incubated in primary antibodies (ChAT 1:100, Millipore, AB144P, c-fos 1:300, Abcam, ab214672; CaMKIIα 1:300, GeneTex, GTX127939; GAD67 1:300, Millipore, MAB5406) in 5% BSA in PBST overnight at 4℃. After washing 3 times with PBST, sections were incubated in fluorophore-conjugated secondary antibodies (1:500, Invitrogen) for 1 h at 37℃. After another 3 washes in PBST, the sections were incubated with DAPI (Beyotime Biotechnology) and mounted on glass slides with a fluorescence mounting medium. Fluorescent signals were imaged with a confocal laser scanning microscope (LSM 780, Zeiss, Germany). The number of fluorescent cells was counted by ImageJ 1.48v (National Institutes of Health, USA).
6
Transcardial Perfusion and Cryosectioning
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After a minimum of 6 weeks of recovery, the animals were euthanized following AVMA guidelines. The animals were perfused transcardially with heparinized saline followed by a solution of 4% or paraformaldehyde in 0.1M phosphate buffer. For each case, the brain was removed and cryoprotected through an ascending series of glycerol solutions. The cryoprotected tissue was then frozen in isopentane and serially sectioned (at 40 µm) using a sledge microtome. Series with every tenth section (400 µm apart) of free-floating sections were processed for either histochemistry using the ABC Elite Kit (Vector laboratories PK-6100) or via immuno-visualization of ChAT, AChE, NeuN, and CFP or mCherry reporters. Primary Antibodies: anti-CFP rabbit polyclonal (Abcam ab290), goat polyclonal anti-ChAT (Millipore AB144P), rabbit polyclonal anti-mCherry (Abcam ab167453), goat anti-mouse Alexa 647 (Invitrogen A21235), mouse anti-AChE (Ivitrogen MA3-042). Secondary Antibodies: biotinylated anti-rabbit (Vector laboratories BA1000), goat anti-mouse Alexa 647 (Invitrogen A21235), goat anti-rabbit Alexa 555 (Invitrogen A21428), goat anti-chicken Alexa 488 (Invitrogen A11039), and goat anti-rabbit Alexa 488 (Invitrogen A11008).
7
Immunohistochemistry Staining Protocol
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The sections were dewaxed, completely rehydrated and for antigen retrieval boiled in sodium citrate 0.1 M pH 6. The sections were washed in phosphate buffer solution (PBT, Na2HPO4·12H2O 0.8 M, Panreac 141678.1214, NaCl 0.15 M, Panreac 121659.1211 and 0.075% Triton-X100, Sigma X100) and incubated in PBT with 1.5% H2O2 for 30 min to inactivate endogenous peroxidase. After inactivation, tissue was washed in PBT and blocked 1 h in PBT with 0.1% albumin bovine serum (BSA, A2153, Sigma) and 10% lysine 1 M (L5626, Sigma). Next, sections were incubated overnight at room temperature in PBT with 0.1% BSA and 0.01% sodium azide (S2002, Sigma) with different antibodies: αCaspase3 (1:300, Cell Signaling #9661), αChAT (1:100, Chemicon AB144P), αβGalactosidase (1:500, Abcam ab9361) and αTH (1:1000, Inst.J.Boy 28020234). The day after, the tissue was rinsed in PBT and incubated 1 h with the appropriate biotinylated secondary antibody at 1:200. Afterwards, the sections were washed in PBT and incubated in PBT with Avidin–Biotin Complex (1:500, Vectastain PK-4000) for 1 h. Finally, tissue was washed in PBT and Tris 0.1 M pH 7 and the immunolabeling was revealed in Tris 0.1 M with 1% 3-3’ diaminobenzidine tetrahydroc (DAB, Acros Organics W0572M) and 0.003% H2O2 leading to a brown precipitate.
8
Neurochemical Mapping of Diencephalic Nuclei
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Mice were anesthetized with isoflurane and perfused transcardially with PBS. Brains were incubated in 4% paraformaldehyde overnight at 4 °C. The rostral to caudal extension of the DMH was cut in 50 μm coronal sections with a vibratome. Sections were blocked with 5% bovine serum albumin at room temperature and then incubated with anti-ChAT (1:1000; Millipore, AB144P), anti-c-fos (1:500; Abcam, ab7963), anti-dsRed (1:1000, Clontech, 632496), anti-cholera toxin subunit b (CTb) (1:500; Abcam, ab62429), and anti-5-HT (1:500; Immunostar, 20080) antibodies diluted in 0.5% Triton X-100 in PBS overnight at 4 °C. For ChAT staining, colchicine (1 μl of 10 mg/ml; Santa Cruz Biotech) was injected into the lateral ventricle of animals 1 day before sacrifice. After incubation in primary antibodies, sections were washed 3 times in PBS and then incubated with Alexa 488 anti-goat IgG (1:500, A11055), Alexa 568 anti-rabbit IgG (1:500, A10042), Alexa 488 anti-rabbit IgG (1:500, A11008), and Alexa 568 anti-mouse IgG for CTb staining (1:500, A11004; Life Technologies) diluted in 0.5% Triton X-100 in PBS for 3 h at room temperature. Tissues were then washed in PBS, dried and mounted with VECTASHIELD mounting media. Images were acquired using a scanning confocal microscope.
9
Multicolor Fluorescent Immunostaining Protocol
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For fluorescent staining, primary antibody were as followed : Tyrosine Hydroxylase (TH, raised in Rabbit, 1:1000, AB152, Sigma-Aldrich), TH (raised in chicken, 1:1000, AB76442, Abcam), Choline acetyltransferase (ChAT, raised in goat, 1:1000, AB144P, Abcam), Alpha synuclein phosphorylated on serine 129 (pSer129, raised in Rabbit, 1:500, AB51253, Abcam), apolipoprotein E (ApoE, raised in goat, 1:500, 178479, Millipore).
Secondary antibody were as follow: 488-Anti Rabbit (raised in Donkey, 1:1000, A2206 , Jackson Immunoresearch); CY3-Anti Rabbit (raised in Goat, 1:1000, A11011, Thermofisher); CY5-Anti Rabbit (raised in Goat, 1:1000, A21244, Thermofisher); 488- Anti Chicken (raised in Goat, 1:1000, A11039, Thermofisher); CY3-Anti Chicken (raised in Donkey, 1:1000, A78951, Jackson Immunoresearch); CY5-Anti Chicken (raised in Donkey, 1:1000, A21449, Jackson Immunoresearch); CY3-Anti Goat (raised in Donkey, 1:1000, A11058, Jackson Immunoresearch); CY5-Anti Goat (raised in Donkey, 1:1000, A214447, Jackson Immunoresearch); 488- Anti Mouse (raised in Donkey, 1:1000, a21202, Thermofisher); CY3-Anti Mouse (raised in Donkey, 1:1000,715–175-150, Jackson Immunoresearch); CY5-Anti Mouse (raised in Donkey, 1:1000, 715–165-150, Jackson Immunoresearch); Biotin-conjugated goat Anti-Rabbit (1:500, B-6648, Sigma Aldrich).
Secondary antibody were as follow: 488-Anti Rabbit (raised in Donkey, 1:1000, A2206 , Jackson Immunoresearch); CY3-Anti Rabbit (raised in Goat, 1:1000, A11011, Thermofisher); CY5-Anti Rabbit (raised in Goat, 1:1000, A21244, Thermofisher); 488- Anti Chicken (raised in Goat, 1:1000, A11039, Thermofisher); CY3-Anti Chicken (raised in Donkey, 1:1000, A78951, Jackson Immunoresearch); CY5-Anti Chicken (raised in Donkey, 1:1000, A21449, Jackson Immunoresearch); CY3-Anti Goat (raised in Donkey, 1:1000, A11058, Jackson Immunoresearch); CY5-Anti Goat (raised in Donkey, 1:1000, A214447, Jackson Immunoresearch); 488- Anti Mouse (raised in Donkey, 1:1000, a21202, Thermofisher); CY3-Anti Mouse (raised in Donkey, 1:1000,715–175-150, Jackson Immunoresearch); CY5-Anti Mouse (raised in Donkey, 1:1000, 715–165-150, Jackson Immunoresearch); Biotin-conjugated goat Anti-Rabbit (1:500, B-6648, Sigma Aldrich).
10
Immunofluorescence for Myocyte and Neuron Characterization
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Immunofluorescence was performed to confirm myocyte and sympathetic neuron phenotypes and to ensure the absence of cholinergic neurons. Cultures were fixed in 4% paraformaldehyde for 10 minutes before being permeabilised and blocked for one hour using a solution containing 10% goat serum, 0.3% bovine serum albumin and 0.1% Triton X in PBS. Primary antibodies to sarcomeric alpha actinin (myocyte marker, A7811, 1:650) and tyrosine hydroxylase (sympathetic neuron marker, ab152, Abcam, 1:250) and choline acetyl transferase (cholinergic neuron marker, ab144P, Abcam, 1:500) were incubated over night at 4 °C. Alexafluor conjugated antibodies were incubated for two hours before the coverslips were washed and mounted using a DAPI containing soft mount Vectashield (Vectorlabs). Imaging was done using a Live Cell Olympus confocal microscope.
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