The following reagents were used: Streptozotocin (Merck, Darmstadt, Germany); AdipoRon (Selleckchem, Houston, TX, USA); a triglyceride assay kit and a non-esterified free fatty acid assay kit (Jiancheng Bioengineering Institute, Nanjing, China); and dihydroethidium (Thermofisher, MA, USA). The following antibodies were used: Rabbit monoclonal to AdipoR1 (EPR6626,Abcam, Cambridge, UK); rabbit polyclonal to RNA-binding protein with multiple splicing (RBPMS; Abcam); rabbit monoclonal to glutamine synthetase (EPR13022(B),Abcam); chicken polyclonal to glial fibrillary acidic protein (GFAP; Abcam); rabbit polyclonal to EGR4 (Abcam); phospho-AMPKα rabbit monoclonal antibody (mAb) and AMPKα rabbit mAb (40H9/D5A2,Cell signaling, Boston, USA); phospho-acetyl CoA carboxylase (ACC) rabbit mAb and ACC rabbit mAb (D7D11/C83B10,Cell signaling); cleaved caspase-3 rabbit mAb (Cell signaling), β-actin (8H10D10,Cell signaling); donkey anti-rabbit AlexaFluor-594 and donkey anti-chicken Alexa 488 (Jackson ImmunoResearch, Philadelphia, USA). The following reagents were used: 5,6-chloromethyl-2ʹ,7ʹ-dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Thermofisher); RNAimax (Thermofisher); Opti-MEM I (Thermofisher); small interfering RNA (siRNA; GenePharma, Shanghai, China); Cell Counting Kit-8 (CCK-8; Dojindo, Tokyo, Japan); and 4ʹ,6-diamidino-2-phenylindole (DAPI; Absin, Shanghai, China).
Donkey anti chicken alexa 488
Manufactured by Jackson ImmunoResearch
Sourced in United States
Donkey anti-chicken Alexa 488 is a secondary antibody that binds to chicken primary antibodies. It is conjugated with the Alexa Fluor 488 fluorescent dye, which emits green fluorescence when excited by a suitable light source. This product can be used in various immunodetection techniques, such as Western blotting, immunohistochemistry, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 594 donkey anti mouse, by Jackson ImmunoResearch (2 mentions)
Lipidtox, by Thermo Fisher Scientific (2 mentions)
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Streptozotocin (stz), by Merck Group (1 mentions)
Dihydroethidium, by Thermo Fisher Scientific (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Donkey anti rabbit alexafluor 594, by Jackson ImmunoResearch (1 mentions)
Opti mem 1, by Thermo Fisher Scientific (1 mentions)
Adiporon, by Selleck Chemicals (1 mentions)
Non esterified free fatty acid assay kit, by Nanjing Jiancheng (1 mentions)
Rbpms, by Abcam (1 mentions)
Cleaved caspase 3 rabbit mab, by Cell Signaling Technology (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Triglyceride assay kit, by Nanjing Jiancheng (1 mentions)
Ab40673, by Abcam (1 mentions)
Chicken anti tyrosine hydroxylase, by Aves Labs (1 mentions)
Goat anti rabbit alexa568, by Thermo Fisher Scientific (1 mentions)
Donkey anti rabbit hrp, by Thermo Fisher Scientific (1 mentions)
Mouse anti gapdh, by Merck Group (1 mentions)
10 protocols using donkey anti chicken alexa 488
1
Adiponectin Receptor Signaling in Diabetic Retinopathy
2
Antibodies and Reagents for Dopamine Analysis
3
Immunostaining of Adult Brains and Fat Bodies
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Immunostaining of adult brains and fat bodies was performed as previously described[25 , 50 (link), 51 (link)]. Tissues were dissected in ice-cold PBS. Brains were fixed overnight in 0.8% paraformaldehyde (PFA) in PBS at 4°C. The fixed brains were washed five times in PBS with 0.5% BSA and 0.5% Triton X-100 (PAT), blocked for 1 hour in PAT + 5% NDS, and then incubated overnight at 4°C with the primary antibodies. Following incubation, the brains were washed five times in PAT, re-blocked for 30 min, then incubated in secondary antibody in block for 4 hr at room temperature. Finally, the brains were washed five times in PAT, then mounted on slides in Slow fade gold antifade. Primary antibodies were as follows: rabbit anti-Dilp5 (1:500; this study); Chicken anti-GFP (1:500; Cat# ab13970, RRID:AB_300798); And Mouse anti-Draper (1:50; DSHB 5D14 RRID:AB_2618105). Secondary antibodies from Jackson ImmunoResearch (1:500) include donkey anti-Chicken Alexa 488 (Cat# 703-545-155, RRID: AB_2340375); donkey anti-mouse Alexa 594 (Cat# 715-585-150, RRID:AB_2340854). Lipid droplets were stained with lipidtox (1:500, Thermo Fisher Cat#H34477) overnight at 4C.
4
Neuronal Synaptic Protein Analysis
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Primary antibodies included: mouse anti-GluA1 (NeuroMAB; 1:10 IHC), rabbit anti-GluA1 (Cell Signaling Technologies; 13185, 1:1,000), mouse anti-GluA2 (NeuroMAB; 1:10 IHC), mouse anti-GluA2 (MAB1189; 1:500), chicken anti-Homer (SySy; 160006, 1:500), mouse anti-SynGAP (Thermo Fisher Scientific; PA1-046, 1:500 IHC, 1:5,000 WB), mouse anti-PSD95 (Thermo Fisher; MA1-045, 1:1,000 WB), rabbit anti-Synaptophysin (PA1-1043, 1:5,000 WB), mouse anti-Cdk5 (Invitrogen; AHZ0492, 1:500), mouse anti-WAVE1 (mAb K91/361; NeuroMab), goat anti-PPA2c (Santa Cruz; sc-6110), rabbit anti-CaMKIIα (Abcam; EP1829Y, 1:1,000), rabbit-anti-p-Synapsin1-Ser 551 (Abcam; Ab32532), rabbit anti-p35 (Cell Signaling; 2680, 1:250), mouse anti-GAPDH (Millipore; MAB374, 1:4,000), and Phalloidin Alexa 647 (Thermo Fisher Scientific; A22287, 1:200). Secondary antibodies included: donkey anti-mouse Alexa 488 (Thermo Fisher Scientific; R37114, 1:200), donkey anti-mouse Dy 488 (Abcam; 96875, 1:500), donkey anti-chicken Alexa 488 (Jackson ImmunoResearch; 703-545-155), and LI-COR (anti-mouse, anti-goat, and anti-rabbit IRDye 680 and 800; 1:10,000).
5
Immunostaining of Mouse Brain Sections
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Mice were perfused with ice cold phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. Brains were incubated in PFA overnight at 4°C, rinsed twice with PBS and then dehydrated for 48 hr in 30% sucrose in PBS at 4°C. The brains were frozen in O.C.T. compound (VWR) and sliced at the cryostat (Leica, Germany). Free-floating sections were rinsed in PBS and then incubated in blocking solution (1% bovine serum albumin and 0.3% Triton X-100 in PBS) containing primary antibodies for 24 hr at 4°C. Sections were washed with PBS three times and incubated for 24 hr at 4°C in blocking solution with secondary antibodies. Immuno-labeled sections were washed three times with PBS and mounted on glass slides. Antibodies used in this study were chicken anti-GFP (Thermo Scientific, A10262, 1:1000), rabbit anti-RFP (Rockland, 600-401-379, 1:2000), donkey anti-chicken alexa 488 (Jackson ImmunoResearch, 703-545-155, 1:1000), donkey anti-rabbit Cy3 (Jackson ImmunoResearch, 711-165-152, 1:1000) and rabbit anti-cleaved caspase-3 (NEB, #9661, 1:500).
6
Immunostaining of GFP-expressing Neurons
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Mice were perfused with 0.1 M PBS followed by 4% paraformaldehyde (PFA). Brains were immersed in PBS-azide overnight. Brains were dissected with a vibratome (Leica) at 100 mm into PBS-azide for mitochondria. Brain sections were blocked in 3% normal donkey serum with 0.3% Triton-X for 30 min at room temperature. Sections were then incubated overnight at 4 °C in primary antibody (1:1500 chicken anti-GFP; Aves Labs, Cat#GFP-1020) diluted in 3% NDS and 0.3% tween 20 solution. On the second day, tissue sections were rinsed in PBS three times for 30 min per rinse followed by rinsing with PBS every hour at room temperature for 8 h, then incubated overnight at 4 °C in secondary antibody, 1:500 donkey anti-chicken-Alexa488 (Jackson ImmunoResearch cat# 703–545-155) diluted in PBS. After secondary incubation, sections were rinsed in PBS every hour for 8 h. Slices were mounted, dried overnight in the dark, and cover slipped with mounting media (Aqua-Polymount; Polysciences, Inc.). DsRed was not amplified via immunostaining, as endogenous virus expression was sufficient for imaging.
7
Immunohistochemical Characterization of Neuronal and Glial Markers
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Primary antibodies used were rabbit anti-HSV-1 (B0114, Dako), chicken anti-NeuN (ab134014, Abcam), mouse anti-NeuN (clone A60, Millipore), and rabbit anti-beta III Tubulin (ab18207, Abcam), rabbit anti-Iba1 (Wako), rabbit anti-STAT1α91 (M-23, Santa Cruz Biotechnology). The mouse anti-A5 antibody (FE-A5), developed by Bruce A. Fenderson at Thomas Jefferson University, was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA. Secondary antibodies used were goat-anti-mouse/rabbit Alexa 555, goat-anti-mouse/rabbit Alexa 488 (Invitrogen), and donkey-anti-chicken Alexa488 (Jackson ImmunoResearch Laboratories). Isolectin B4 conjugated to FITC (Sigma) was added to cultures at a concentration of 10μg/mL to stain KH10 neurons in chambers [67 ]. Counterstaining was done by incubation with DAPI (Invitrogen). Samples were mounted using FluorSave Reagent (Calbiochem). When indicated, cells were treated with 12.5U/mL (unless noted) IFNβ (PBL Interferon Source), 100ng/mL IFNγ (Miltenyi Biotec) or 100ng/mL IFNλ2 (PeproTech) for 18 hours prior to infection or for the specified amount of time prior to staining.
8
Immunostaining for Adult Drosophila Brains and Fat Bodies
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Immunostaining of adult brains and fat bodies was performed as previously described [65 (link),66 (link)]. Tissues were dissected in ice-cold PBS. Brains were fixed overnight in 0.8% paraformaldehyde (PFA) in PBS at 4°C. The fixed brains were washed 5 times in PBS with 0.5% BSA and 0.5% Triton X-100 (PAT), blocked for 1 h in PAT + 5% NDS, and then incubated overnight at 4°C with the primary antibodies. Following incubation, the brains were washed 5 times in PAT, re-blocked for 30 min, and then incubated in secondary antibody in block for 4 h at room temperature. Finally, the brains were washed 5 times in PAT, then mounted on slides in Slow fade gold antifade. Primary antibodies were as follows: rabbit anti-Dilp5 (1:500; this study), Chicken anti-GFP (1:500; Cat# ab13970, RRID:AB_300798), and Mouse anti-Draper (1:50; DSHB 5D14 RRID:AB_2618105). Secondary antibodies from Jackson ImmunoResearch (1:500) include donkey anti-Chicken Alexa 488 (Cat# 703-545-155, RRID: AB_2340375) and donkey anti-mouse Alexa 594 (Cat# 715-585-150, RRID:AB_2340854). Lipid droplets were stained with LipidTox (1:500, Thermo Fisher Cat#H34477) overnight at 4°C.
9
Cre-Expressing Neurons Visualization
10
Immunohistochemistry of Serotonin Neurons
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Immunohistochemistry was performed on brains collected from ChR2+ and ChR2− mice (see Supplementary Methods 3.1 ). Primary antibodies used were mouse monoclonal anti-TPH2 (1:500; Sigma-Aldrich) and chicken anti-GFP (1:1000; Abcam). Secondary antibodies used were donkey anti-mouse Alexa 594 (1:1000; Invitrogen) and donkey anti-chicken Alexa 488 (1:1000; Jackson Immunoresearch). Sections were imaged using a confocal microscope.
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