Mice were sacrificed by cervical dislocation. Their brains were removed and fixed in 4% paraformaldehyde (PFA) overnight. After dehydration, the tissue was embedded in paraffin and coronally sectioned (7 µm) on a rotary microtome (Leica, RM45). Select sections from the corresponding region of the locus ceruleus (LC), dorsal raphe nuclei (DR), hippocampus, or frontal cortex in mutant and control mice were incubated overnight at 4 °C with primary anti-CREB (1:100, Abcam, United Kingdom, cat no. ab32515), anti-NeuN (1:100, Millipore, USA, cat. no. MAB377), anti-Tph2 (1:100, Millipore, USA, cat. no. AB1541) and anti-TH (1:500, Millipore, USA, cat. no. AB1542) antibodies. Antigen-bound primary antibodies were visualized with anti-rabbit Alexa-488, anti-sheep Alexa-594, and anti-mouse Alexa-594 (Invitrogen, USA) coupled secondary antibodies. Stained sections were analyzed and acquired under a fluorescence microscope (Nikon Eclipse50i, Japan) equipped with a camera and specialized software (NIS Elements, ver. BR 3.0).
Anti mouse alexa594
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-mouse Alexa594 is a fluorescent labeling reagent that is specifically designed to detect mouse-derived proteins or antibodies in various biological applications. It is a red-fluorescent dye that can be conjugated to mouse-specific probes, allowing for the visualization and localization of target molecules in samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit alexa488, by Thermo Fisher Scientific (9 mentions)
Alexa 488 anti rabbit, by Thermo Fisher Scientific (7 mentions)
Anti rabbit alexa594, by Thermo Fisher Scientific (6 mentions)
Anti mouse alexa 488, by Thermo Fisher Scientific (5 mentions)
Anti rabbit alexa647, by Thermo Fisher Scientific (3 mentions)
Axiovert 100m microscope, by Zeiss (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Formalin solution, by Merck Group (2 mentions)
Cyclin d1, by Cell Signaling Technology (2 mentions)
Antibodies to β actin, by Merck Group (2 mentions)
Fluorescein lotus tetragonolobus lectin, by Vector Laboratories (2 mentions)
Anti rabbit hrp and anti mouse hrp secondary antibodies, by Vector Laboratories (2 mentions)
Anti rabbit dylight 488, by Vector Laboratories (2 mentions)
Phospho 4ebp1, by Cell Signaling Technology (2 mentions)
Cyclin e1, by Cell Signaling Technology (2 mentions)
Rapamycin, by LC Laboratories (2 mentions)
Anti rat alexa 488, by Thermo Fisher Scientific (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
31 protocols using anti mouse alexa594
1
Immunohistochemical Analysis of Neuronal Markers
2
Immunofluorescence Characterization of Cardiomyocytes
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Cells grown on chamber slides (BD) were fixed (paraformaldehyde 4% in PBS, 15 min). Every following step was performed in PBS containing 1% BSA (Sigma). Cells were permeabilised (0.1% Triton X-100, 25 min) and blocked with 5% normal goat serum (NGS, 15 min). Primary antibody incubation was performed at 4°C overnight, dilution 1 : 200. Primary antibodies, anti-HuNu (Millipore), anti-cTnT (Abcam), anti-Mef2c (New England Biolabs), anti-connexin 43 (Abcam), and anti-alpha-sarcomeric actinin (Abcam), were washed and samples were incubated with secondary antibodies (1 : 500) for 30 minutes at RT. Secondary antibodies used were as follows: anti-rabbit-Alexa488, anti-mouse-Alexa594, and anti-rabbit-Alexa555 (Invitrogen) and anti-mouse Alexa555 (Cell Signaling).
Stained specimens were kept in mounting media (PermaFluor, ThermoScientific) and images were acquired using a fluorescent microscope (EVOS, Life Technologies) or spinning disk confocal microscope (Quorum Zeiss AxioVert, SickKids Imaging Facility, Toronto). Images were quantified using Volocity™ (Perkin-Elmer) imaging software.
Stained specimens were kept in mounting media (PermaFluor, ThermoScientific) and images were acquired using a fluorescent microscope (EVOS, Life Technologies) or spinning disk confocal microscope (Quorum Zeiss AxioVert, SickKids Imaging Facility, Toronto). Images were quantified using Volocity™ (Perkin-Elmer) imaging software.
3
Immunostaining of Drosophila Adult Brains
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Fly crosses were set under 12:12 LD cycles at 25C. Flies eclosing within 24 hours were collected and kept under their respective conditions until the ages indicated in each experiment. Adult brains were dissected in PBS (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4 and 1.8mM KH2PO4) within 10 minutes. Then brains were fixed in 3.7% formalin solution for 30 minutes. Brains were washed with 0.3% PBSTx for 4 times before primary antibody incubation. Primary antibodies were diluted in 0.3% PBSTx with 5% normal goat serum and incubation was done at 4C overnight. Brains were washed for 4 times with 0.3% PBSTx after primary antibody incubation. Secondary antibodies were diluted in 0.3% PBSTx with 5% normal goat serum and incubation was done at 4C overnight. Primary antibody dilutions were done as the followings: mouse anti-PDF (1:800, DSHB), rabbit anti-PDF (1:1000, from Nitabach Lab), mouse anti-GFP (1:1000). Secondary antibody dilutions were done as the followings: anti-mouse Alexa594 (1:800, invitrogen), anti-mouse Alexa488 (1:800, invitrogen), anti-rabbit Alexa594 (1:800, invitrogen), anti-rabbit Alexa488 (1:800, invitrogen), anti-rabbit Alexa647 (1:800, invitrogen).
4
Immunofluorescence Imaging of Toxoplasma Infection
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MEF cells were infected with Toxoplasma for 24h and then were fixed with 4% paraformaldehyde for 20 minutes and blocked with PBS supplemented with 2% BSA. Cells were permeabilized in blocking buffer containing 0.1% Triton X-100 for 30 min then incubated with rabbit anti-CAT1 (Abcam) and mouse anti-SAG1 (Invitrogen) for 1 hour. Alternatively, the cells were incubated with anti-CAT1 without permeabilization, followed by incubation with anti-SAG1. Secondary goat anti-rabbit Alexa-fluor 488 and anti-mouse Alexa-594 (Invitrogen) was applied for 1 hour followed by Vectashield mounting media. DAPI was used as a co-stain to visualize host and parasite nuclei (Vector Labs). Images were acquired with Leica inverted DMI6000B microscope with 63x oil immersion objective.
5
Drosophila Circadian Rhythm Immunohistochemistry
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Fly crosses were set on the standard cornmeal-yeast-agar medium under 12:12 or 10:10 (for the misalignment experiment) LD cycles at 25°C. Flies eclosing within 24 hours were collected and kept under the conditions they were raised until the ages indicated in each experiment. Adult brains were dissected in PBS (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4and 1.8mM KH2PO4) within 10 minutes. Then brains were fixed in 3.7% formalin solution (diluted from 37% formalin solution, Sigma-Aldrich) for 30 minutes. Brains were washed with 0.3% PBSTx (PBS with 0.3% Triton-X) for 4 times before primary antibody incubation. Primary antibodies were diluted in 0.3% PBSTx with 5% normal goat serum and incubation was done at 4°C overnight. Brains were washed for 4 times with 0.3% PBSTx after primary antibody incubation. Secondary antibodies were diluted in 0.3% PBSTx with 5% normal goat serum and incubation was done at 4°C overnight. Primary antibody dilutions were done as the followings: mouse anti-PDF (1:800, DSHB), rabbit anti-PER (1:8000, from Rosbash Lab). Secondary antibody dilutions were done as the followings: anti-mouse Alexa594 (1:800, invitrogen), anti-mouse Alexa488 (1:800, invitrogen), anti-rabbit Alexa594 (1:800, invitrogen), anti-rabbit Alexa488 (1:800, invitrogen), anti-rabbit Alexa647 (1:800, invitrogen).
6
Molecular Markers in Kidney Disease
7
Molecular Markers in Kidney Disease
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Antibodies against total rpS6, phospho-rpS6, Cyclin D1, Cyclin E1, and phospho-4E-BP1 were from Cell Signaling Technology (Beverly, MA). Fluorescein Lotus-tetragonolobus Lectin (LTL) was purchased from Vector Laboratories Inc, (Burlingame, CA). Anti-synaptopodin antibody was from Acris Antibodies GmbH (San Diego, CA). Anti-rabbit HRP and anti-mouse HRP secondary antibodies, anti-rabbit dyLight 488 were from Vector laboratories (Burlingame, CA). Anti-mouse Alexa 594 was from Invitrogen Life Technologies (Carlsbad, CA). Rapamycin was purchased from LC Laboratories (Woburn, MA). Antibodies to β-actin and other chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO).
8
Immunostaining for Circadian Proteins
9
Immunofluorescence Staining and Mitophagy Quantification
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Cells were seeded in the confocal dishes with different treatments, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 diluted in PBS. Next, the cells were blocked with 2.5% bovine serum albumin (BSA) in PBS for 20 min. Primary antibodies against VZV gE (SC-56995), GM130 (BD Biosciences; 610822), anti-HA (51064-2-AP), and anti-MYC (16286-1-AP) were diluted in 0.5% BSA in PBS and incubated with cells for 1 h, followed by secondary antibody incubation using anti-mouse Alexa 594 (Invitrogen; A11005), anti-mouse FITC (Invitrogen; 31569) and anti-rabbit Alexa 488 (Invitrogen; A11008). The cells were then stained with 4ʹ,6-diamidino-2-phenylindole (DAPI).
To measure mitolysosome formation, the cells were treated with media containing MitoTracker Green (Invitrogen) and LysoTracker Deep Red (Invitrogen) to visualize mitochondria and lysosomes. After 30 min, the cells were imaged using a confocal microscope (LSM900; Carl Zeiss) and the percentage of cells with mitolysosomes was quantified by counting more than 100 cells. The quantification of mitophagy was assessed by measuring pH-dependent fluorescence using HeLa-mtKeima cells, as previously described [16 (link), 24 (link)].
To measure mitolysosome formation, the cells were treated with media containing MitoTracker Green (Invitrogen) and LysoTracker Deep Red (Invitrogen) to visualize mitochondria and lysosomes. After 30 min, the cells were imaged using a confocal microscope (LSM900; Carl Zeiss) and the percentage of cells with mitolysosomes was quantified by counting more than 100 cells. The quantification of mitophagy was assessed by measuring pH-dependent fluorescence using HeLa-mtKeima cells, as previously described [16 (link), 24 (link)].
10
Drosophila Circadian Rhythm Immunohistochemistry
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