Immunohistochemistry was performed as described previously37 (link). Briefly, 10 μm thick cryosections were fixed in 4% paraformaldehyde for 20 min at room temperature and then incubated in 0.1% Triton X100/phosphate-buffered saline (PBS) for 20 min at 37 °C followed by incubation in 3% bovine serum albumin/PBS for 1 h at room temperature. The cryosections were then incubated with the following primary antibodies for 1 day at 4 °C: rabbit polyclonal antibody against α7-nAChR (sc-5544, 1:20; Santa Cruz Biotechnology), rabbit polyclonal antibody against GAD65/GAD67 (ab11070, 1:200; Abcam), rabbit polyclonal antibody against GABA (A2052, 1:100; Sigma, St. Louis, MO, USA), and goat polyclonal antibody against Brn3a (sc-31984, 1:40; Santa Cruz Biotechnology). The secondary antibodies (all from Invitrogen-Molecular Probes, Eugene, OR, USA) were Alexa Fluor 555-conjugated donkey anti-rabbit IgG antibody (A31572, 1:1,000), Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (A11070, 1:500), and Alexa Fluor 488-conjugated donkey anti-goat IgG antibody (A11055, 1:500). The sections were finally counterstained with the nucleic acid stain Hoechst 33258 (H3569, 1:2,000; Invitrogen-Molecular Probes) in PBS and imaged using a laser scanning confocal microscope (TCS SP8; Leica Microsystems, Heidelberg, Germany).
Ab11070
Manufactured by Abcam
Sourced in United States, Germany
Ab11070 is a primary antibody specific for the detection of a target protein. The antibody is produced in a host species and is affinity-purified from the host serum. The antibody is supplied in a buffer solution and is ready-to-use for various applications, such as Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Nbp1 88283, by Novus Biologicals (2 mentions)
A11070, by Thermo Fisher Scientific (1 mentions)
Tcs sp8, by Leica (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
A11055, by Thermo Fisher Scientific (1 mentions)
A2052, by Merck Group (1 mentions)
Sc 31984, by Santa Cruz Biotechnology (1 mentions)
Ab78078, by Abcam (1 mentions)
Ab1218, by Abcam (1 mentions)
Bs 0230r, by Bioss Antibodies (1 mentions)
Alexa 488, by Jackson ImmunoResearch (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Lsm 800 laser, by Zeiss (1 mentions)
Ab109902, by Abcam (1 mentions)
Infinite m1000 pro, by Tecan (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Immuno blot pvdf membrane, by Bio-Rad (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Fv1200, by Olympus (1 mentions)
Ab13970, by Abcam (1 mentions)
10 protocols using ab11070
1
Immunohistochemistry of Neuronal Markers
2
Immunohistochemical Analysis of Embryo Markers
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Analyses of embryos and explants were analyzed using immunohistochemistry according to standard whole-mount or cryostat sectioning techniques [33 (link)]. In this study, antibodies used were: anti-TUJ1 (1:1000 dilution; ab 78078, Abcam, Cambridge, UK); anti-FGF3 (produced by ABclonal, Wuhan, China; 1:200); anti-GAD65/67 (1:1000 dilution; ab11070, Abcam, Cambridge, UK); anti-parvalbumin (1:200 dilution; PV, bs-1299R, Bioss, Beijing, China); anti-FGFR1 (1:200 dilution; bs-0230R, Bioss, Beijing, China); and anti-green fluorescent protein (GFP) mouse monoclonal (ab1218, Abcam, Cambridge, Uk). Secondary antibodies were conjugated to Alexa 647 or Alexa 488 (1:1000 dilution; Jackson, Lancaster, PA, USA); FGFR1 antibody blocking peptide (bs-0230P, Bioss; 50 ng/μL); and FGF3 antibody blocking peptide (bs-1255P, Beijing, China, Bioss; 50 ng/μL).
3
Immunofluorescent Labeling of Zebrafish Brains
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Larvae were fixed in 4% para-formaldehyde/PBS overnight at 4 °C. They were then rinsed in PBS. The brains were dissected out, and permeabilized using 1% BSA (fraction V; Sigma), 0.1% DMSO and 0.1% Triton X-100. The anti-GAD65/67 (Abcam ab11070, RRID:AB_297722; 1:500) has been previously used in zebrafish [70 (link)]. The brains were incubated in the primary antibody overnight, rinsed several times in PBS, then incubated in secondary antibody (Alexa 488 goat anti-rabbit; 1:1000). After washing, these were mounted in 1.2% agarose/PBS. Imaging was carried out using a Zeiss LSM 800 laser scanning confocal microscope, with a 40× water immersion objective.
4
Measuring Mitochondrial PDH Activity
5
Western Blot Analysis of GAD65/67 and Actin
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Whole embryos were lysed in modified RIPA buffer containing 150 mM sodium chloride, 50 mM Tris (pH 7.4), 1% Triton X-100, 1% sodium deoxycholate, 1% SDS, and proteasome inhibitor with PMSF, then centrifuged at 4°C for 15 minutes. Equal amounts of protein in each sample were resolved by SDS-PAGE on a 10% Mini-PROTEAN TGX Precast Gel (Bio-Rad) and transferred to an Immuno-Blot PVDF membrane (Bio-Rad). The membrane was blocked with 5% (w/v) non-fat milk and incubated with anti-GAD65/67 (ab11070, Abcam) and anti-actin (R1207-1, Huabio) antibodies. After washing three times for 10 minutes each with Tris-buffered saline containing Tween-20, the membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Finally, the membranes were incubated in chemiluminescence reagent (34080, Pierce) and exposed to X-ray film.
6
Immunohistochemical Labeling of GAD65/67 in Fish Brains
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Larvae were fixed in 4% paraformaldehyde in phosphate‐buffered saline (PBS) overnight and then rinsed several times with PBS. After peeling the skin off the brains, samples were incubated in 1%BSA and then overnight at 4 degrees in primary antibody (anti‐GAD65/67, Abcam ab11070, 1:500 (Cheng et al., 2017 )). The following day, the brains were washed several times with PBS and then incubated in secondary antibody (Alexa 488 goat anti‐rabbit, 1:1000) overnight at 4˚C. After multiple washes with PBS, brains were mounted in low‐melting agarose and imaged on a laser scanning confocal microscope (Zeiss LSM800) using a 40x water‐dipping objective. A total of 7 fish were imaged.
7
Localization of GAD65/67 in Transgenic Zebrafish
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Tg(gad1b:EGFP) embryos at 4 dpf were fixed by 4% PFA and cryo-sectioned with 20 µm width. The slices were sequentially treated with 1× PBS, Antigen Retrieval Solution (Beyotime, P0090), and 5% BSA. The slices were then incubated with chicken-anti-GFP (Abcam, ab13970, 1:5,000 in 5% BSA/PBS) and rabbit-anti-gad65/67 (Abcam, ab11070; 1:500 in 5% BSA/PBS) at 4°C for >16 h, washed with 1× PBS, incubated with Alexa Fluor 594 goat anti-rabbit and Alexa Fluor 488 donkey anti-chicken for 3 h at room temperature. The slices were washed with 1× PBS, air dried, and mounted with coverslip. The signals were checked on a confocal microscope (Olympus, FV1200) under 60× (water, NA = 1.20) objective. Rabbit anti-gad65/67 was omitted in the control group. The conclusion was drawn based on two independent replicates.
8
Western Blot Analysis of Retinal Proteins
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Western blotting was performed according to our previously described methods37 (link). Briefly, retinal lysates were centrifuged at 12,000 × g for 10 min at 4 °C. Ten micrograms of each sample was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrotransferred onto polyvinyldifluoridine membranes (Immobilon-P; Millipore, Billerica, MA, USA). The membranes were then blocked with 5% nonfat milk for 2 h at room temperature and incubated with the following primary antibodies overnight at 4 °C: goat polyclonal antibody against α7-nAChR (sc-1447, 1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit polyclonal antibody against GAD65/GAD67 (ab11070, 1:1000; Abcam, Cambridge, MA, USA). The membranes were incubated with horseradish peroxidase-conjugated rabbit anti-goat immunoglobulin (Ig)G (305-035-003, 1:20000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and horseradish peroxidase-conjugated goat anti-rabbit IgG (sc-2004, 1:4,000; Santa Cruz Biotechnology). The relative intensities of the protein bands were quantified by scanning densitometry using Image J software (National Institutes of Health, Bethesda, MD, USA). β-actin was used as an internal standard.
9
Protein Expression Analysis by SDS-PAGE and Western Blot
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For SDS-PAGE, 11% polyacrylamide gels were freshly prepared, followed by Western blot on nitrocellulose membranes (GE Healthcare, Little Chalfont, UK). Membranes were blocked for 1 h with 5% BSA in TBS-T (TBS with 1% Tween 20). Primary antibodies were incubated overnight at 4°C.
GAD and PDXK proteins were detected with the GAD67/65 specific antibody (ab11070 1:1,000, abcam, Berlin, Germany) and PDXK specific antibody (NBP1-88283, 1:1000, novusBio, Wiesbaden, Germany). β-Actin (GTX26276, 1:5,000, GeneTex/Biozol, Irvine, CA, USA) served as loading control.
Signals were detected using the ECL plus system (GE Healthcare, Little Chalfont, UK).
GAD and PDXK proteins were detected with the GAD67/65 specific antibody (ab11070 1:1,000, abcam, Berlin, Germany) and PDXK specific antibody (NBP1-88283, 1:1000, novusBio, Wiesbaden, Germany). β-Actin (GTX26276, 1:5,000, GeneTex/Biozol, Irvine, CA, USA) served as loading control.
Signals were detected using the ECL plus system (GE Healthcare, Little Chalfont, UK).
10
Evaluating Artemisinin's Impact on Hippocampal Neurons
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DIV14 primary hippocampal neurons were incubated for 2 h with corresponding DMSO/artemisinin concentrations (1 µM, 3 µM, 10 µM). Neurons were fixed in 4% paraformaldehyde in PBS for 15 min.
After washing twice with PBS, 50 mM NH4Cl was added for 10 min. Blocking with 5% goat serum in PBS (permeabilized with 0.2% Triton X-100) for 30 min at 22°C followed. Primary antibodies were incubated for 1 h in blocking solution without Triton X-100. GAD, gephyrin and PDXK proteins were detected with the GAD67/65 specific antibody (ab11070 1:500, abcam, Berlin, Germany), gephyrin specific antibody (147111, 1:500, Synaptic Systems, Göttingen, Germany) and PDXK specific antibody (NBP1-88283, 1:150, novusBio, Wiesbaden, Germany), respectively. Secondary antibodies gαmCy3, gαrCy5 (1:500; Dianova, Hamburg, Germany) and ActinGreenTM (R37110, Thermo Fisher Scientific, Irvine, CA, USA) were applied for 1 h. Cells were stained with 4',6-diamino-2-phenylindole (DAPI) and slides were mounted with Mowiol.
After washing twice with PBS, 50 mM NH4Cl was added for 10 min. Blocking with 5% goat serum in PBS (permeabilized with 0.2% Triton X-100) for 30 min at 22°C followed. Primary antibodies were incubated for 1 h in blocking solution without Triton X-100. GAD, gephyrin and PDXK proteins were detected with the GAD67/65 specific antibody (ab11070 1:500, abcam, Berlin, Germany), gephyrin specific antibody (147111, 1:500, Synaptic Systems, Göttingen, Germany) and PDXK specific antibody (NBP1-88283, 1:150, novusBio, Wiesbaden, Germany), respectively. Secondary antibodies gαmCy3, gαrCy5 (1:500; Dianova, Hamburg, Germany) and ActinGreenTM (R37110, Thermo Fisher Scientific, Irvine, CA, USA) were applied for 1 h. Cells were stained with 4',6-diamino-2-phenylindole (DAPI) and slides were mounted with Mowiol.
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