Brain sections were washed in 0.1 M PBS, blocked in 3% normal donkey serum/0.25% Triton X-100 in PBS for 1 hour at room temperature and then incubated overnight at room temperature in blocking solution containing primary antiserum. Primary antibodies used were Rat anti-mCherry 1:1,000 (Life Technologies), Rabbit anti-GFP 1:1,000 (Life Technologies), 1:1000 Rabbit anti-CRF (Thermo-Fisher), 1:1,000, Mouse anti-Tyrosine Hydroxylase (TH) LNC1 1:1,000 (EMD Millipore), Goat anti-Choline Acetyltransferase (ChAT)1:1000 (EMD Millipore), Rabbit anti-Calcitonin gene-related peptide (CGRP) 1:1000 (Peninsula), Rabbit anti-FOS 1:1,000 (EMD Millipore), Goat-anti Cholera Toxin B (CTB) 1:1,000 (List Technologies). The next morning sections were washed 3X PBS and then incubated in Alexa Fluor Donkey secondary antibody 1:1000 (Molecular Probes) diluted in blocking solution for 2 h at room temperature. The ChAT antibody also requires an antigen retrieval step: 30min incubation at 60°C in sodium citrate buffer prior to primary antibody incubation. Sections were washed 3× in PBS, mounted onto gelatin subbed slides, and cover-slipped with DAPI-Vectashield mounting media (Vector Labs).
Goat anti choline acetyltransferase chat
Manufactured by Merck Group
Sourced in Germany, United States, United Kingdom
Goat anti-choline acetyltransferase (ChAT) is a laboratory reagent used in research applications. ChAT is an enzyme involved in the synthesis of the neurotransmitter acetylcholine. This antibody can be used to detect and study the expression and distribution of ChAT in various biological samples.
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Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (2 mentions)
Rabbit anti gfap, by Agilent Technologies (2 mentions)
Rat anti cd68, by Bio-Rad (2 mentions)
Neuronal nuclei (neun), by Merck Group (2 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (2 mentions)
Rabbit anti tyrosine hydroxylase th, by Merck Group (2 mentions)
Neuronal nuclei (neun), by Vector Laboratories (2 mentions)
P tauser202 thr205, by Thermo Fisher Scientific (2 mentions)
Rat anti mouse b220 cd45r biotinylated, by BD (2 mentions)
Abc vector elite, by Vector Laboratories (2 mentions)
Vectashield dapi mounting media, by Vector Laboratories (1 mentions)
Rat anti mcherry, by Thermo Fisher Scientific (1 mentions)
Rabbit anti fos, by Merck Group (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Axiocam 2, by Zeiss (1 mentions)
Axiovision acquisition software, by Zeiss (1 mentions)
Cytoseal 60, by Electron Microscopy Sciences (1 mentions)
Lsm 510 meta, by Zeiss (1 mentions)
Cm1950, by Leica (1 mentions)
15 protocols using goat anti choline acetyltransferase chat
1
Immunohistochemical Labeling of Brain Sections
2
Immunohistochemical Staining of Mouse Spinal Cord
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As previously described [43 (link)] for the spinal cord, mice were anaesthetized and transcardially perfused with 4% PFA in PBS. The lumbar spinal cord was removed, post-fixed in 4% PFA for 2 h, incubated in 30% sucrose in PBS, embedded in OCT, flash-frozen, and cut at 18 µm thickness. The sections were then rinsed for 5 min with PBS and incubated for 2 h at room temperature in blocking solution (PBS, 5% donkey serum, 0.3% Triton-X100, 0.05% Tween-20). This was followed by overnight incubation at +4 °C with the following primary antibodies: rabbit anti-TGFβ-R1 (Sigma-Aldrich, Saint-Louis, MO, USA, SAB4502958, 1:200) and goat anti-choline acetyl transferase (ChAT) (Merck, Darmstadt, Germany, AB144P, 1:100). Sections were washed and incubated for 1 h at room temperature with appropriate AlexaFluor-conjugated secondary antibodies (ThermoFisher Scientifc, Waltham, MA, USA). Slides were mounted in Mowiol solution. Image acquisition was carried out on a Zeiss Axio imager Z2-module Apotome 2.0 (Car Zeiss AG, Oberkochen, Germany).
3
Immunohistochemical Analysis of Brainstem Neurons
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Rats were sacrificed by overdose of urethane and transcardially perfused with saline followed by cold (4°C) paraformaldehyde (PFA; 4%). The medulla was harvested and postfixed in 4% PFA overnight at 4°C, then cryoprotected in sucrose (30%) in standard PBS (1–3 days at 4°C). PBS contained (mM): NaCl 137, KCl 2.7, Na2HPO4 10, KH2PO4 1.8, pH 7.4. Brainstems were transversely sectioned at 40 μm. Free-floating sections were incubated overnight in PBS containing 0.1% Triton X-100 (PBT) and mouse anti-NeuN primary antibody (1:500; EMD Millipore) or goat anti-cholineacetyl transferase (ChAT; 1:100; EMD Millipore). The tissue was washed in PBS, 6 times for 5 minutes per wash, and then incubated separately for 2–4 hours in a solution of PBT containing either donkey anti-mouse Alexa Fluor 647 secondary antibody (1:250; Jackson ImmunoResearch Laboratories, Inc.) or donkey anti-goat Alexa Fluor 488 (1:250; Invitrogen), for NeuN and ChAT, respectively. The tissue was washed in PBS, 6 times for 5 minutes. Slices were mounted onto polylysine-coated slides, dehydrated overnight at 22°C, and coverslipped using Cytoseal 60 (Electron Microscopy Sciences). Slides were analyzed using a fluorescent microscope with AxioVision acquisition software (AxioCam2, Zeiss).
4
Immunohistochemical Analysis of Cholinergic Neurons
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Mice were deeply anesthetized and perfused transcardially with 0.1 m phosphate buffer (PB) followed by ice-cold 4% paraformaldehyde. Coronal sections of the striatum were cut at 30 μm on a cryostat microtome (Leica CM1950) in antifreeze buffer (1:1:3 volume ratio of ethyl glycerol, glycerol, and 0.1 m PB) and stored at −20°C before further analysis. The sections were pre-incubated in 5% normal horse serum and 0.3% Triton X-100 in 0.1 m PB for 40 min after washing steps, and incubated overnight at 4°C with the primary antibodies [goat anti-choline acetyltransferase (ChAT), 1:100; Millipore; RRID:AB_262156 )]. On the second day, sections were incubated with fluorophore-conjugated species-specific secondary antibodies (donkey anti-goat, 1:1000; Abcam) for 2 h at room temperature. Brain sections were rinsed in PBS and directly coverslipped by fluorescent mounting medium (VECTASHIELD, Vector Laboratories). Multi-labeling fluorescent immunostainings of juxtacellularly filled neurons were analyzed using a laser-scanning microscope (LSM 510 Meta, Zeiss) using 20×/0.3 NA interference contrast lens (20× zoom).
5
Immunohistochemical Profiling of Neuronal Markers
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Immunohistochemistry was carried out using the following antibodies: mouse anti-Calbindin (CB, 1:3000, Sigma), rabbit anti-Calretinin (CR, 1:400, Invitrogen), mouse anti-Reelin (Reln, G10, 1:2000, provided by AMG), rabbit anti-Pax6 (1:200, Covance), mouse anti-neurofilament (NF; 2H3,1:500, Hybridoma bank), mouse anti-Isl1 (39.4D5, 1:500, Hybridoma bank), rabbit anti-Isl1 (1:2000, Abcam), goat-anti cleaved Caspase3 (1:500, bcam), goat-anti-choline acetyltransferase (ChAT, 1:100, Millipore) and rat-anti Fzd3 (1:400, R&D). Cortical barrels were studied in vibratome sections of flattened cortex, stained with guinea pig anti-Vglut2 (1:2000, Millipore). Signal was detected with an anti-mouse/rabbit universal ABC kit (PK-6200, Universal, Vector) or the following fluorescent secondary antibodies: donkey anti-mouse Alexa fluor 546 (1:1000, Invitrogen), donkey anti-mouse Alexa fluor 488 (1:1000, Invitrogen), donkey anti-rabbit Alexa fluor 488 (1:1000, Invitrogen), donkey anti-rabbit Alexa fluor 546 (1:1000, Invitrogen), goat anti-guinea pig Alexa fluor488(1:1000, Invitrogen).
6
Immunofluorescence Staining of Spinal Cord Tissue
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After blocking with 5% bovine serum albumin (BSA) for 1 h, the spinal cord tissue slices were incubated overnight at room temperature (RT) with different primary antibodies: goat anti-choline acetyltransferase (ChAT; 1:1000; Millipore), rabbit anti-ionized calcium binding adaptor molecule 1 (Iba1; 1:1000, Wako), mouse anti-glial fibrillary acidic protein (GFAP; 1:1000, Sigma), mouse anti-neuronal nitric oxide synthases (nNOS; 1:1000, Invitrogen), and rabbit anti-low-affinity nerve growth factor receptor (p75; 1:1000, Millipore). After repeated washes in 0.01 M phosphate-buffered saline (PBS, pH 7.4), slices were incubated with corresponding secondary antibodies conjugated with Alexa Fluor-488 or 594 (1:1000, Invitrogen) for 4 h at room temperature in the dark. Besides, sections of biceps were stained with α-Bungarotoxin (α-BTX) antibody (1:1000; Alexa Fluor 488 conjugated; Invitrogen) for 2 h at room temperature in the dark. Following thorough washes, fluorescence mounting medium (Dako, Copenhagen, Denmark) was used to mount tissue onto coverslips for microscopic examination. Finally, fluorescent images were captured using a Zeiss fluorescence microscope (Zeiss, Gottingen, Germany) equipped with an ORCA-Flash 4.0 v2 digital CMOS camera (Hamamatsu Photonics, Iwata City, Japan).
7
Immunohistochemical Co-labeling Protocol
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Free-floating sections were incubated for 1 hr in a blocking solution (PBS containing 0.1% Triton X-100 and 5% BSA). The tissue was then incubated overnight at room temperature in primary antibodies: goat anti-choline acetyl transferase [ChAT; 1:100; Millipore, Burlington, MA, USA] alone for co-labelling with GCaMP6, or rabbit anti-tryptophan hydroxylase [TPH; 1:500; Sigma-Aldrich, St Louis, MO, USA] alone for co-labelling with GCaMP6.
Slices were washed in PBS (6 × 5 min) and then incubated in a blocking solution to which secondary antibodies were added; donkey anti-rabbit Alexa Fluor 594 (1:250; Jackson Laboratory, Bar Harbor, ME, USA) for co-labelling with GCaMP6, or donkey anti-goat Alexa Fluor 594 (1:250; Jackson Laboratory, Bar Harbor, ME, USA; RTN) for co-labelling with GCaMP6 in RTN tissue or donkey anti-goat Alexa Fluor 568 (1:250; Jackson Laboratory, Bar Harbor, ME, USA) for co-labelling with GCaMP6 in pFL tissue. The tissue was then incubated 2–4 hr at room temperature. Tissue was washed in PBS (6 × 5 min). Slices were mounted on polysine adhesion slides and were coverslipped with Vectashield Antifade Mounting Medium with DAPI (Vectorlabs, Burlingame, CA, United States).
Slices were washed in PBS (6 × 5 min) and then incubated in a blocking solution to which secondary antibodies were added; donkey anti-rabbit Alexa Fluor 594 (1:250; Jackson Laboratory, Bar Harbor, ME, USA) for co-labelling with GCaMP6, or donkey anti-goat Alexa Fluor 594 (1:250; Jackson Laboratory, Bar Harbor, ME, USA; RTN) for co-labelling with GCaMP6 in RTN tissue or donkey anti-goat Alexa Fluor 568 (1:250; Jackson Laboratory, Bar Harbor, ME, USA) for co-labelling with GCaMP6 in pFL tissue. The tissue was then incubated 2–4 hr at room temperature. Tissue was washed in PBS (6 × 5 min). Slices were mounted on polysine adhesion slides and were coverslipped with Vectashield Antifade Mounting Medium with DAPI (Vectorlabs, Burlingame, CA, United States).
8
Immunohistochemical Localization of Steroid Receptors in Murine Brain
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Brain sections from male mice were washed with PBS and blocked for 1 hr at room temperature with 2% bovine serum albumin (BSA) containing 0.3% Triton X-100 in PBS. They were then incubated for 48 hr at 4°C with the following primary antibodies: rabbit anti-AR (1:200; Epitomics ) or rabbit anti-ERα (1:5000; Millipore Corp) and goat anti-choline acetyltransferase (ChAT) (1:1000; Millipore Corp) or mouse anti-calcitonin gene-related peptide (CGRP) (1:8000; Abcam, Cambridge, UK). After the primary antibody was removed by rinsing, sections were incubated with secondary antibodies for 1 hr at room temperature . The following secondary antibodies were used for double-staining: anti-rabbit Alexa Fluor (AF) 488 (1:1000; Invitrogen, Grand Island, NY, USA) and anti-goat AF 546 (1:1000; Invitrogen) or anti-mouse AF 546 (1:1000; Invitrogen).
9
Immunolabeling of Retinal Cell Subtypes
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Fixed retinas were either frozen and sectioned at 20 μm in a cryostat or stained as whole mounts. Retina sections or whole mounts were incubated in PBS with 3% donkey serum and 0.3% Triton X-100 for blocking, followed by primary antibodies for ≥24 h at 4°C and secondary antibodies for ∼4 h. Retinas were then washed with PBS and mounted in Fluoromount G (Southern Biotech).
Primary antibodies used in this study were: rabbit anti-GFP (1:1000, Millipore); rabbit and mouse anti-calbindin (1:2000, Swant and 1:100, Swant); goat anti-choline acetyltransferase (ChAT), (1:400, Millipore); mouse anti-Brn3a (1:1000, Millipore); goat anti-Chx10 (1:200, Santa Cruz); mouse anti-AP2 (1:1000, DSHB); mouse anti-HCN4 (1: 1000, clone N114.10); rabbit anti-PKC alpha (1:1000, P4334 in Sigma); rabbit anti-secretagogin (1:4000, Biovendor); rabbit anti-arrestin (1:200, Milipore); rabbit anti-glutamine synthetase (1:1000, BD Biosciences); rabbit anti-sox9 (1:1000, Chemicon); mouse anti-connexin 43 (1:50, Life Sciences); rabbit antibody to Dab1 (a kind gift from B. Howell, SUNY Upstate Medical University). Secondary antibodies were conjugated to DyLight 649 (Jackson ImmunoResearch), Alexa Fluor 568, or Alexa Fluor 488 (Invitrogen) and used at 1:500.
Primary antibodies used in this study were: rabbit anti-GFP (1:1000, Millipore); rabbit and mouse anti-calbindin (1:2000, Swant and 1:100, Swant); goat anti-choline acetyltransferase (ChAT), (1:400, Millipore); mouse anti-Brn3a (1:1000, Millipore); goat anti-Chx10 (1:200, Santa Cruz); mouse anti-AP2 (1:1000, DSHB); mouse anti-HCN4 (1: 1000, clone N114.10); rabbit anti-PKC alpha (1:1000, P4334 in Sigma); rabbit anti-secretagogin (1:4000, Biovendor); rabbit anti-arrestin (1:200, Milipore); rabbit anti-glutamine synthetase (1:1000, BD Biosciences); rabbit anti-sox9 (1:1000, Chemicon); mouse anti-connexin 43 (1:50, Life Sciences); rabbit antibody to Dab1 (a kind gift from B. Howell, SUNY Upstate Medical University). Secondary antibodies were conjugated to DyLight 649 (Jackson ImmunoResearch), Alexa Fluor 568, or Alexa Fluor 488 (Invitrogen) and used at 1:500.
10
Antibody Characterization for Neurodegeneration
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Antibodies used in the study were: rabbit anti-spatacsin (Protein Tech, directed against aa 2094–2443 of human spatacsin); rabbit anti-spastizin (Murmu et al., 2011 (link)); mouse anti-α-tubulin (Abcam); mouse anti-NeuN (Millipore); rabbit anti-GFAP (DAKO); monoclonal mouse anti-Calbindin, 1:300, (Swant); goat anti-Choline acetyltransferase (ChAT), 1:500, (Millipore); rabbit anti-Neurofilament-L, 1:100, (Millipore); mouse Alexa Fluor® 488 conjugated anti-Synaptophysin, 1:25, (Millipore); rat anti-Lamp1 (Clone 1D4B), mouse anti-p62 (Abcam); rabbit anti-LC3 (Novus Biologicals); goat anti-caspase-3 (Millipore). For immunoblotting, the secondary antibodies were conjugated to horseradish peroxidase (Jackson Laboratories) or fluorochromes (IR-dye 800 or IR-dye 680; LI-COR). Secondary antibodies used for immunofluorescence were purchased from Life Technologies.
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