For immunostainings, adult males were directly collected in fixation solution (4% paraformaldehyde in PBS (10 mM Na2HPO4, 2 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl) supplemented with 0.1% Triton X-100). Dissected brains were fixed for 30 min at room temperature, then washed in PBT (PBS plus 0.3% Triton X-100, used for all washing steps) before blocking in PBT supplemented with 5% normal goat serum for 2 h. Incubation with the following primary antibodies was done overnight at 4°C: mouse anti-phospho-Y214-SGG (1:400; clone 5G2F12, Papadopoulou et al., 2004 (link)), rabbit anti-ITP (1:10000, Hermann et al., 2013 (link)), mouse anti-PDF (1:1500, clone C7, Developmental Studies Hybridoma Bank, Iowa City, IA, United States), rabbit anti-PER (1:750, Stanewsky et al., 1997 (link)) and chicken anti-GFP (1:750; Millipore, Upstate, Temecula, CA, United States). Secondary antibodies were AlexaFluor 488, Cy3 or Cy5-conjugated and were purchased from Molecular Probes (Eugene, OR, United States) and Dianova (Hamburg, DE). Embedding of brains was done in Vectashield (Vector Laboratories, Burlingame, CA, United States) and confocal images were collected with identical settings for all genotypes with a Leica SP5 microscope (Leica Microsystems, Wetzlar, DE). Image processing was carried out in an identical manner with the ImageJ distribution Fiji (Schindelin et al., 2012 (link)).
Chicken anti gfp
Manufactured by Merck Group
Sourced in United States, Japan
Chicken anti-GFP is a laboratory reagent used to detect and quantify the presence of green fluorescent protein (GFP) in biological samples. It is an antibody raised in chickens that specifically binds to GFP, allowing researchers to visualize and measure GFP-tagged proteins or cells.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Mouse anti actin, by Merck Group (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Mouse anti flag, by Merck Group (2 mentions)
Sp5 microscope, by Leica (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Mouse anti pdf, by Developmental Studies Hybridoma Bank (1 mentions)
Mouse anti gad67, by Merck Group (1 mentions)
Rabbit anti shank1a, by Merck Group (1 mentions)
Rabbit anti gad65, by Merck Group (1 mentions)
Rabbit anti gad67, by Merck Group (1 mentions)
Rabbit anti erk, by Santa Cruz Biotechnology (1 mentions)
Mouse anti pax6, by Abcam (1 mentions)
Rabbit anti gfp, by Abcam (1 mentions)
Rabbit anti notch1, by Abcam (1 mentions)
Mouse anti gapdh, by Abcam (1 mentions)
Mouse anti satb2, by Abcam (1 mentions)
Rabbit anti tbr2, by Abcam (1 mentions)
25 protocols using chicken anti gfp
1
Immunostaining of Adult Drosophila Brains
2
Immunohistochemistry of Spinal Cord Cells
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Immunohistochemistry on 12 μm thick cryostat sections of lumbar level (L) 4 to 5 spinal cord was performed as previously described (Betley et al., 2009 (link)). Rabbit anti-βgal (gift from J. Sanes) (Gray and Sanes, 1991 (link)), rabbit anti-ChAT (generously provided by S.B.-M. and T.M.J., unpublished data), rabbit anti-GAD65 1:50,000 (Betley et al., 2009 (link)), mouse anti-GAD67 1:10,000 (Millipore), rabbit anti-GAD67 1:10,000 (Betley et al., 2009 (link)), chicken anti-GFP 1:1,000 (Millipore), sheep anti-GFP 1:1,000 (Molecular Probes), rat anti-NB2 (1A6) 1:4 (Shimoda et al., 2012 (link)), chicken anti-Pv 1:10,000 (generously provided by S.B.-M. and T.M.J., unpublished data), rabbit anti-RFP (Rockland), rabbit anti-Shank1a 1:64,000 (Betley et al., 2009 (link)), rabbit anti-Shank1a 1:1,000 (Millipore), mouse anti-Syt1 1:100 (ASV48, Developmental Studies Hybridoma Bank), and guinea pig anti-vGluT1 1:32,000 (Betley et al., 2009 (link)).
3
Comprehensive Antibody Immunostaining Protocol
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The primary antibodies used were mouse anti-GFP (Invitrogen—1:1000), rabbit anti-GFP (Abcam—1:5000), chicken anti-GFP (Millipore—1:1000), mouse anti-SatB2 (Abcam—1:400), mouse anti-βIII-tubulin (Covance—1:1000), rabbit anti-βIII-tubulin (Covance—1:1000), rabbit anti-Pax6 (Covance—1:2000), mouse anti-Pax6 (Abcam—1:250), rabbit anti-GAPDH (Sigma—1:1000), rabbit anti-Tbr2 (Abcam—1:500), rabbit anti-Erk (Santa Cruz Biotechnology—1:1000), rabbit anti-Notch1 (Abcam—1:250), rabbit anti-RFP (MBL—1:1000), mouse anti-Actin (Sigma—1:2000), mouse anti-MG-H1 (Cell Biolabs—1:50), mouse anti-GAPDH (Abcam—1:1000), rabbit anti-GLO1 (Abcam 1:500), mouse anti-puromycin (Kerafast—1:1000). The donkey anti-mouse and anti-rabbit Alexa 488, 555 and 647-conjugated secondary antibodies were obtained from ThermoFisher and used at 1:500 dilutions. HRP-conjugated goat anti-mouse, anti-rabbit or anti-chicken secondary antibodies were purchased from ThermoFisher and used at 1:5000 dilutions. NIR-conjugated goat anti-mouse and anti-rabbit secondary antibodies were acquired from LI-COR and used at 1:25,000 dilutions.
4
Immunohistochemical Analysis of pS6 in Brain
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After transcardial perfusion fixation with 4% paraformaldehyde/PBS, brains were sliced transversely (50 μm thick) with microtome and processed for immunohistochemistry. Primary antibodies, including goat anti-GFP (Rockland, 1:1000) and chicken anti-GFP (Millipore, 1:1000) were used. Immunofluorescence was performed with a combination of Alexa Fluor 488- or Alexa Fluor 594-labeled anti-goat, anti-chicken, or anti-rabbit secondary antibodies (1:250) and 4ʹ,6ʹ-diaminodino-2-phenylindole (DAPI, 1:5000). For analysis of pS6 levels, primary antibodies against pS6-Ser235/236 (rabbit, 1:1000, Cell Signaling) were used. Effective immunostaining of pS6 required an antigen retrieval protocol as previously described [58 (link)]. Briefly, sections were incubated in target retrieval solution (DAKO) in 85°C for 20 minutes followed by washing with PBS for t3 times before the incubation with primary antibody.
5
Immunohistochemical Analysis of the Mouse Olfactory Bulb
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We perfused mice transcardially with PBS followed by 4% paraformaldehyde, and soaked the brains in 30% sucrose. We sectioned OBs coronally (30 µm) on a sliding microtome and performed immunohistochemsitry on floating sections in 5% heat-inactivated goat serum and 0.5% triton in PBS. We used the following primary antibodies: rabbit anti-calretinin (CR) (1:2000) and mouse anti-calbindin (CB) (1:1000) (Swant, Bellinzona, Switzerland), mouse anti-Tyrosine Hydroxylase (TH) (1:500) (Immunostar, Hudson, WI, USA), mouse anti-Tbr2 (1:100) (Abcam, Bristol, UK) and mouse anti-NeuN (Millipore, Billerica, MA, USA). GCaMP was amplified using chicken anti-GFP (1:1000, Millipore). The following secondary antibodies were used: biotinylated goat anti-rabbit (1:500), DyLight549 conjugated goat anti-mouse (1:500) and DyLight488 conjugated goat anti-chicken, (Jackson ImmunoResearch, West Grove, PA, USA). Amplification was carried out using Cy5 conjugated streptavidin (Jackson ImmunoResearch). Slices were imaged with a SP50 confocal microscope, via a 60X (1.4 NA) oil objective (Leica, Wetzlar, Germany). Counting of neuronal somata was carried out manually using ImageJ.
6
Labeling Excitatory Neurons with GCaMP6s
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Mice were deeply anesthetized and transcardially perfused with 9.25% w/v sucrose in distilled water followed by 4% PFA in PBS. Brains were then dissected out and post-fixed in 4% PFA for one week at 4°C. Coronal sections (50 μm) were cut on a microtome (Thermo Fisher Scientific) and were kept free-floating at 4°C until further processing. Immunohistochemistry was carried out using the primary antibodies chicken anti-GFP (1:1000; Millipore) labeling GCaMP6s and rabbit anti-Homer3 (1:250; Synaptic Systems), which labels excitatory neurons. After washing, sections were incubated with species-specific secondary antibodies conjugated to Alexa Fluor 488 (1:200, Life Technologies) or Cy3 (1:200, Life Technologies) and mounted with mounting medium containing DAPI (Vectashield).
Images were acquired using a laser-scanning confocal microscope (Leica, TCS SP8), across serial optical sections (spaced at 1 μm) acquired with a 20x objective (0.75 NA) at a resolution of 1024 x 1024 with a sequential scan using excitation lasers for DAPI (405 nm), Alexa488 (488 nm) and Cy3 (561 nm). Quantitative analysis was performed with the “Cell Counter” plug-in for ImageJ, by counting GFP expressing cells among Homer 3 expressing cells in cortical layer 2/3 (100–300 μm from the pial surface).
Images were acquired using a laser-scanning confocal microscope (Leica, TCS SP8), across serial optical sections (spaced at 1 μm) acquired with a 20x objective (0.75 NA) at a resolution of 1024 x 1024 with a sequential scan using excitation lasers for DAPI (405 nm), Alexa488 (488 nm) and Cy3 (561 nm). Quantitative analysis was performed with the “Cell Counter” plug-in for ImageJ, by counting GFP expressing cells among Homer 3 expressing cells in cortical layer 2/3 (100–300 μm from the pial surface).
7
Immunofluorescence Staining of Cellular Structures
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After washing in PBS, sections were treated with PBS—0.1%, Triton X-100—10% serum for 30 min. They were then incubated overnight at 4 °C with the following primary antibodies diluted in the blocking buffer: mouse anti-acetylated tubulin 1:100 (Sigma); mouse anti-α-tubulin (1:100, Sigma-Aldrich); rabbit anti-Cenpj: 1:200 (Proteintech); rabbit anti-Cdk5rap2 (1:100, Milipore); rabbit anti-centrin: 1:100 (Milipore); chicken anti-GFP (1:700, Millipore); rabbit anti-pH3 (1:200, Upstate); mouse anti-γ-tubulin (1:100, Abcam) and mouse anti-tyrosinated tubulin 1:100 (Sigma). Sections were then incubated with appropriate fluorescent secondary antibodies. To detect Cenpj, a step of antigen retrieval with citrate was performed before the blocking (sodium citrate pH=6 for 20 min at 90 °C).
8
Immunohistochemical Analysis of Rat Brain
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Adult rat brains were perfused with cold 4% paraformaldehyde. Embryonic brains (E10.5-E18.5) from TH-GFP rats were immersion fixed in 4% paraformaldehyde overnight. Brains were washed with 1xPBS and transferred to 30% sucrose solution in 1xPBS and mounted in O.C.T. for sectioning. Adult brains were sectioned at 30 µm on a Leica freezing microtome and embryonic brains of various stages were sectioned at 15 µm on a Microm cryostat as described in [12] (link). Sections were processed with primary antibodies (rabbit-anti-TH 1∶200, Pel Freeze, chicken-anti-GFP 1∶200, Millipore) at 4°C overnight using procedures for immunocytochemistry as described previously [13] (link). All secondary antibodies were AlexaFluor antibodies from Invitrogen used at 1∶200 for 30 min at room temperature. Slides were covered with ProLong Gold antifade reagent (Invitrogen). Cultures were rinsed twice and then fixed with 4% paraformaldehyde and stained with primary antibodies at 4°C overnight as previously detailed [14] (link). Cultures were also counter stained with Dapi (Molecular Probes) at 1∶1000. Stained cells and sections were examined using an OlympusIX81 Image Analysis System.
9
Comprehensive Immunostaining Protocol
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Primary antibodies: mouse IgG2a anti-AnkyrinG (clone N106/36; 1:100), mouse IgG2b anti-AnkyrinG (clone N106/65; 1:75), and mouse IgG1 anti-Caspr (1:100), all from Neuromab; mouse IgG1 anti-Pan Nav (clone K58/35; 1:150; Sigma); mouse anti-Calbindin (1:500; Sigma), rabbit anti-Calbindin (1:300; Swant), rabbit anti-Caspr (1:300; Abcam), rat anti-PLP (1:10; kindly provided by Dr. K. Ikenaka, Okasaki, Japan), mouse IgG2b anti-MBP (1:200; SMI99, Sigma), rabbit IgG anti-Iba1 (1:500; Wako), chicken anti-GFAP (1:500; Aves Labs), rat anti-PDGFrα (1:100; BD Biosciences), rabbit IgG anti-TMEM119 (1:100; Sigma), rabbit IgG anti-P2Y12r (1:300; Alomone, human tissue), rabbit anti-P2Y12R (1:300; Anaspec, mouse tissue), chicken anti-GFP (1:250; Millipore), mouse IgG2a anti-iNOS (1:100; BD Biosciences), goat anti-IGF1 (1:50; R&D System), and chicken anti-mCherry (1:1000; Abcam). Secondary antibodies corresponded to goat or donkey anti-chicken, goat, mouse IgG2a, IgG2b, IgG1, rabbit and rat coupled to Alexa Fluor 488, 594, 647, or 405 from Invitrogen (1:500), or goat anti-mouse IgG1 DyLight from Jackson Immuno Research (1:600).
10
Immunostaining of Neural Progenitor Markers
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Dissected tissues were fixed 5 min or more in 4% formaldehyde/PBS depending on the primary antibody. Stainings were performed in 0.5% triton/PBS with antibody incubations separated by several washes. Tissues were then transferred in Vectashield (Clinisciences, France) with or without DAPI for image acquisition. Primary antibodies were: chicken anti-GFP (1:1000, Tebubio, France), mouse anti-Mira (1:50, A. Gould), guinea-pig anti-Mira (1:1000, A. Wodarz), guinea-pig anti-Asense (1:1000, J. Knoblich), rabbit anti-PH3 (1:500, Millipore, Billerica, MA), rat anti-PH3 (1:500, Abcam, UK), rat anti-Elav (1:50, DSHB, Iowa City, IA), rat anti-Chinmo (1:500, N. Sokol), rabbit anti-Castor (1:500, W. Odenwald), guinea-pig anti-Hunchback (1:500, J. Reinitz), guinea-pig anti-Kruppel (1:500, J. Reinitz), rabbit anti-Pdm (1:500, X. Yang), mouse anti-Svp (1:50, DSHB), rabbit anti-Imp (1:500, P. Macdonald), rat anti-Lin-28 (1:500, N. Sokol). Adequate combinations of secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were used to reveal expression patterns.
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