The following primary antibodies were obtained commercially and used at the indicated dilutions: mouse anti-Nestin (1:200, R&D Systems, Minneapolis, MN; MAB2736), mouse anti-NeuN (1:500; Millipore, Etobicoke, ON; MAB377), rabbit anti-ShcB (1:1000; Santa Cruz, CA; sc-33,808). Rabbit anti-ShcC (1:2000) was kindly provided by Dr. John O’Bryan (Medical University of South Carolina, Charleston, SC). Rabbit anti-ShcD (1:1000) was generated against the unique CH2 region. Its specificity has been confirmed by preadsorption with the original antigen prior to immunohistochemistry [8 (link), 13 (link)], by immunoblot against lysates prepared from cells expressing other Shc proteins [8 (link)], and by immunoblot on brain lysates prepared from ShcD knockout mice [32 (link)].
Mab377
Manufactured by Santa Cruz Biotechnology
Sourced in United States
MAB377 is a monoclonal antibody product offered by Santa Cruz Biotechnology. It is designed for use in various research applications. The core function of MAB377 is to serve as a specific detection tool for its target antigen.
Automatically generated - may contain errors
Lab products found in correlation
Neuronal nuclei (neun), by Merck Group (3 mentions)
Lsm 780, by Zeiss (2 mentions)
Mouse anti neun, by Merck Group (1 mentions)
Mouse anti nestin, by R&D Systems (1 mentions)
Confocal laser scanning microscope, by Olympus (1 mentions)
Lsm 700, by Zeiss (1 mentions)
Alexa fluor 488 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Mab377x, by Merck Group (1 mentions)
Ferritin, by Merck Group (1 mentions)
α hemoglobin, by Santa Cruz Biotechnology (1 mentions)
β hemoglobin, by Santa Cruz Biotechnology (1 mentions)
Hemin, by Frontier Scientific (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Vectashield mounting medium with 4 6 diamidino 2 phenylindole dapi, by Vector Laboratories (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Anti il6rα, by Santa Cruz Biotechnology (1 mentions)
Bs 1049r, by Bioss Antibodies (1 mentions)
Tunel staining, by Boster Bio (1 mentions)
Mouse anti βiii tubulin tuj1, by Merck Group (1 mentions)
10 protocols using mab377
1
Antibody Detection Techniques for Shc Proteins
2
Panx1 Expression in Trigeminal Ganglia
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Detection of Panx1 in satellite glia and neurons from trigeminal ganglia was performed as previously described10 (link) using chicken anti-Panx1 (1:500; extracellular loop (CZVQQKSSLQSES); Aves Lab #6358); mouse anti-NeuN (1:100; Millipore - MAB377); goat anti-glutamine synthase (1:200; Santa Cruz - sc-6640), and secondary Alexa conjugated goat anti-chicken, goat anti-mouse, and donkey anti-goat antibodies (1:2000). Images were acquired using an Olympus confocal laser scanning microscope equipped with a 40× water-immersion lens (0.80 NA), and FITC, Texas red and UV filter sets.
3
Immunofluorescent Analysis of Brainstem and Cerebellum
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Ctrl and HCb animals were perfused transcardially with saline followed by 4% paraformaldehyde in PB (0.1 M, pH 7.4) under renewed general anesthesia induced by i.p. injections of sodium pentobarbital (60 mg/kg). Each brain was removed from the skull, post-fixed in the same fixative for 2 h, and then transferred to 30% sucrose in PB at 4 °C until it sank. Brainstem and cerebellum were cut into sections using a freezing microtome and collected in PB.
Sections were incubated overnight with primary antibodies including mouse anti-neuronal nuclei (NeuN; 1 : 200; Millipore, Darmstadt, Germany; MAB-377), rabbit anti-DSCR1 (RCAN1; 1 : 200, Santa Cruz, Dallas, TX, USA; sc-66864) prepared in PB containing 0.3% Triton X-100. Each incubation step was followed by three 5-min rinses in PB. Afterwards, sections were incubated 2 h at RT with secondary antibodies including Alexa Fluor 555 donkey anti-rabbit IgG (1 : 200; Molecular Probes; Eugene, OR, USA; A31572), Alexa Fluor 488 donkey anti-mouse IgG (1 : 200; Molecular Probes, 31571). Sections were examined under a confocal laser-scanning microscope (Zeiss LSM700).
Sections were incubated overnight with primary antibodies including mouse anti-neuronal nuclei (NeuN; 1 : 200; Millipore, Darmstadt, Germany; MAB-377), rabbit anti-DSCR1 (RCAN1; 1 : 200, Santa Cruz, Dallas, TX, USA; sc-66864) prepared in PB containing 0.3% Triton X-100. Each incubation step was followed by three 5-min rinses in PB. Afterwards, sections were incubated 2 h at RT with secondary antibodies including Alexa Fluor 555 donkey anti-rabbit IgG (1 : 200; Molecular Probes; Eugene, OR, USA; A31572), Alexa Fluor 488 donkey anti-mouse IgG (1 : 200; Molecular Probes, 31571). Sections were examined under a confocal laser-scanning microscope (Zeiss LSM700).
4
Antibody Sources and Protein Reagents
5
Immunohistochemical Analysis of Neural Markers
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Ad libitum–fed animals were perfused with 4% paraformaldehyde transcardially. The brains were immersed in 10% (w/v) sucrose and, after 24 hours, transferred to 30% (w/v) sucrose. After that, they were embedded in optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA) and cut into 20-μm coronal sections using a cryostat. The sections were incubated in blocking solution in PBS (0.2% of Triton X-100) and 5% BSA for 2 hours at 21°C and then incubated overnight at 4°C in mouse anti-NeuN (1:300; Millipore, MAB377), anti-IL6Rα (1:200; Santa Cruz Biotechnology, sc660), anti–phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (D13.14.4E) XP Rabbit mAb #4370 (Cell Signaling Technology), and anti–SF-1 antibody (A-1; 1:200; Santa Cruz Biotechnology, sc-393592). After washing in PBS, sections were placed in secondary donkey anti-mouse fluorescein isothiocyanate (1:500; Santa Cruz Biotechnology, sc 2010) and goat anti-rabbit Alexa Fluor 546 (1:500; Thermo Fisher Scientific, CA, USA) for 2 hours and mounted with VECTASHIELD Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI) (#H-1200, Vector Laboratories Inc., Burlingame, CA, USA). The images were acquired with a confocal laser microscope (LSM 780, Zeiss, Jena, Germany). Digital/electronic zoom of highlighted areas was performed when necessary.
6
Immunostaining and TUNEL Assay for Brain Sections
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IF staining was performed on 16-μm-thick frozen brain sections as described previously [53 (link)]. Briefly, the brain sections were permeabilized with 0.35% Triton X-100 for 15 min, blocked for 60 min at 37 °C with goat serum, then incubated with the matching primary antibodies at 4 °C overnight. The primary antibodies included mouse anti-caspase 1 (1:40, Santa Cruz, sc-56036), mouse anti-NeuN (1:200, MilliporeSigma, MAB377), mouse anti-GSDMDC1 (1:50, Santa Cruz, sc-393656), rabbit anti-NeuN (1:100, Proteintech, 26975-1-AP), and rabbit anti-α7nAChR (1:50, Bioss, bs-1049R). On the following day, the slides were incubated with corresponding secondary antibodies and 4′,6-diamidino-2-phenylindole. Subsequently, the slides were imaged using a microscope (Nikon, Tokyo, Japan) and analyzed using the ImageJ software.
TUNEL staining (Boster, Wuhan, China) was used to count the number of dead cells, following the manufacturer’s instructions as previously described [54 (link)]. The number of TUNEL-positive cells was statistically analyzed by a blinded observer using the ImageJ software.
TUNEL staining (Boster, Wuhan, China) was used to count the number of dead cells, following the manufacturer’s instructions as previously described [54 (link)]. The number of TUNEL-positive cells was statistically analyzed by a blinded observer using the ImageJ software.
7
Immunofluorescence Staining of Neural Markers
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Immunofluorescence was performed as described before38 (link),50 (link), briefly: sections were incubated for 48 h at 4 °C in 0.01 M PBS pH 7.4 containing 2% Triton X-100 (TBS), 1:100 normal donkey serum and the primary antibodies. Sections were incubated overnight with appropriate secondary antibodies, extensively washed in TBS and mounted in antifade mounting medium Mowiol (4–88 reagent, Calbiochem 475904). The following primary antibodies and dilutions were used: chicken anti-GFP, 1:000 (AvesLabs, GFP-1020); rabbit, polyclonal anti-GFAP 1:1.500 (Dako Z0334), rabbit anti-hNESTIN, 1:500 (Millipore, ABD69); mouse anti-hNCAM, 1:200 (Santa Cruz, sc-106); mouse anti hNu, 1:1000 (Millipore, MAB128); mouse anti-NeuN, 1:1000 (Chemicon, MAB377); goat anti-SOX10, 1:1000 (Santa Cruz, sc-17342); mouse anti-β-III tubulin (Tuj1), 1:500 (Sigma, T8660). The following secondary antibodies and dilutions were used: donkey anti-rabbit (Cy3 labeled, 1:800, Jackson ImmunoResearch, 711-165-152; donkey anti mouse (Cy3 labeled, 1:800, Jackson ImmunoResearch 715-165-151); donkey anti goat (Cy3 labeled, 1:800, Jackson ImmunoResearch 705-165-147); donkey anti chiken (AlexaFluor488 labeled, 1:400, Jackson ImmunoResearch 703-545-155).
8
Immunohistochemical Analysis of Mouse Brain
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After deep anaesthetization with avertin, mice were transcardially perfused with PBS, followed by 4% PFA. Brains were postfixed in 4% PFA overnight, cryoprotected in 30% sucrose PBS, and cut coronally in 40 μm serial sections with a cryostat. Brain sections that were collected in 24-well plates, were incubated with blocking buffer for 1h at room temperature, and then incubated with primary antibodies at 4 °C overnight. The following primary antibodies were used: anti-GFP (chicken, 1:1000, AVES, #GFP-1010), anti-NeuN (mouse, 1:1000, Millipore, Burlington, MA, USA, #MAB377), and anti-TrkB (rabbit, 1:50, Santa Cruz, #377218). After three washes in PBS, sections were incubated with fluorescent secondary antibodies containing 5 μg/mL Hoechst for 1 h at room temperature. Sections were washed three times by PBS, mounted onto slides in a mounting medium, and coverslipped for imaging with a confocal microscope (Zeiss LSM780).
9
Immunofluorescence Quantification of Parvalbumin Neurons
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Adult mice were sacrificed and perfused with 4% paraformaldehyde and cryostat sections (20 μm) of various brain regions were subjected to immunofluorescence, as described5 (link). Immunofluorescence images were acquired on a Zeiss Z1 fluorescent microscope after Immunostaining using primary antibodies for Parvalbumin (Swant, PV235, ×500), NeuN (Millipore, MAB377, anti-mouse, ×500), and c-Fos (Santa Cruz, sc52, rabbit, ×50), followed by Cy2-, Cy3-conjugated secondary antibodies (Jackson Labs) at ×1000 dilution. For immunofluorescence images, three independent fields at ×20 magnification from six sections were imaged and PV+ cells counted using ImageJ. Investigators were blinded to genotype.
10
Immunohistochemical Analysis of Mouse Brain
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