CV-1 (African green monkey kidney fibroblast, M.L. Nibert own collection, HMS) cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum (HyClone) and 10μg/ml gentamicin (Invitrogen). MRV strains T1L and T3DN were derived from the Bernard N. Fields lab. Rabbit polyclonal antisera specific for μ2 and μNS were used as described previously [23 (link), 46 (link)]. Mouse antibody (MAb) anti-acetylated a-tubulin (clone 6-11B-1) and mouse MAb anti-α-tubulin (clone B-5-1-2) were obtained from Sigma-Aldrich. Rabbit polyclonal anti-tubulin (H-300) and mouse MAb anti-GFP (B-2) were obtained from Santa Cruz Biotechnology, Inc. Rabbit polyclonal anti-GFP (ab290) was obtained from Abcam. Mouse MAb HA.11 was obtained from Covance; goat anti-rabbit immunoglobulin G (IgG) conjugated to Alexa 594, goat anti-mouse IgG conjugated to Alexa 594, and goat anti-mouse IgG conjugated to Alexa 488, were obtained from Molecular Probes, Invitrogen.
Goat anti mouse igg conjugated to alexa 594
Manufactured by Thermo Fisher Scientific
Sourced in United States
Goat anti-mouse IgG conjugated to Alexa 594 is a secondary antibody reagent. It is designed to detect and visualize mouse primary antibodies in various immunoassays and imaging techniques.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse e600 microscope, by Olympus (2 mentions)
Colorview 3 camera, by Olympus (2 mentions)
Cryostar nx70 cryostat, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Rabbit polyclonal anti gfp ab290, by Abcam (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Cell Signaling Technology (1 mentions)
Anti rabbit igg conjugated to alexa 488, by Thermo Fisher Scientific (1 mentions)
Anti α tubulin mouse monoclonal antibody, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Fluorsave, by Merck Group (1 mentions)
Vectashield mounting media, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Ab11024, by Abcam (1 mentions)
Ab9498, by Abcam (1 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (1 mentions)
Ab7817, by Abcam (1 mentions)
Goat anti rabbit igg conjugated to alexa 488, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
6 protocols using goat anti mouse igg conjugated to alexa 594
1
Immunostaining Assay for Mammalian Reovirus Proteins
2
Immunofluorescence Microscopy Assay
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Goat anti-mouse IgG conjugated to Alexa 594 and anti-rabbit IgG conjugated to Alexa 488 antibodies (Molecular Probes Inc., Eugene, OR, USA); mouse anti-α-tubulin monoclonal antibody and 4′,6-diamidino-2-phenylindole (DAPI) (Cell Signalling Technology, Danvers, MA, USA); rabbit anti-Caspase 3 polyclonal antibody, active (cleaved) form and Fluorsave (Merck Millipore, Darmstadt, Germany) were purchased from the indicated commercial sources. The protease inhibitor cocktail and all the remaining reagents were from Sigma-Aldrich (St. Louis, MO, USA).
3
Immunofluorescent Localization of Steroidogenic Enzymes
4
Immunostaining of Vein Sections
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Vein samples were embedded in Frozen Section Medium (Richard-Allan Scientific Neg 50, Thermo Scientific) and frozen in dry ice-cooled isopenthane. Frozen tissue blocks were stored at —80C. The samples were cut in 8 um thick sections on a CryoStar™ NX70 Cryostat (Thermo Scientific™), mounted on SuperFrost Plus Adhesion slides and immediately post-fixed for 30 s in cold 95% ethanol. Slides were stored at —20C awaiting immunostaining.
Sections were immunostained for the presence of CD31 (1:50; Abcam #ab9498), von Willebrand factor (1:500; Abcam #ab6994), VEGFR-2 (1:200; Acris Antibodies TA337222), alfa smooth muscle actin (1:500, Abcam #ab7817), CD41 (1:1000; Abcam #ab11024) and fibrinogen (1:250; Abcam #ab118488). Antibodies were diluted in blocking buffer (5% goat serum/3% BSA in PBS), and slides were incubated at 4 °C overnight. Negative controls were made omitting the primary antibody. The secondary antibodies used were goat anti-rabbit IgG conjugated to Alexa 488 and goat anti-mouse IgG conjugated to Alexa 594 (Life Technologies), used at 1:500. Each immunostaining procedure was performed in triplicate and repeated at least twice per vein sample.
The stained sections were mounted with prolong gold antifade (Invitrogen), containing DAPI for nuclear staining. Imaging was done using an upright Nikon Eclipse E600 microscope equipped with an Olympus ColorView III camera.
Sections were immunostained for the presence of CD31 (1:50; Abcam #ab9498), von Willebrand factor (1:500; Abcam #ab6994), VEGFR-2 (1:200; Acris Antibodies TA337222), alfa smooth muscle actin (1:500, Abcam #ab7817), CD41 (1:1000; Abcam #ab11024) and fibrinogen (1:250; Abcam #ab118488). Antibodies were diluted in blocking buffer (5% goat serum/3% BSA in PBS), and slides were incubated at 4 °C overnight. Negative controls were made omitting the primary antibody. The secondary antibodies used were goat anti-rabbit IgG conjugated to Alexa 488 and goat anti-mouse IgG conjugated to Alexa 594 (Life Technologies), used at 1:500. Each immunostaining procedure was performed in triplicate and repeated at least twice per vein sample.
The stained sections were mounted with prolong gold antifade (Invitrogen), containing DAPI for nuclear staining. Imaging was done using an upright Nikon Eclipse E600 microscope equipped with an Olympus ColorView III camera.
5
Cartilage Histology and Immunostaining
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Cartilage samples were embedded in Frozen Section Medium (Richard-Allan
Scientific Neg50, Thermo Scientific) and frozen in dry ice-cooled isopentane.
Frozen tissue blocks were stored at −80 °C. The samples were cut in 9- to 10-µm
thick sections on a CryoStar NX70 Cryostat (Thermo Scientific), mounted on
SuperFrost Plus Adhesion slides, stored at −80 °C and fixed for 60 seconds in
cold 95% ethanol directly before starting immunostaining. Sections were
immunostained for the presence of type II collagen (COL2; clone II-4C11; MP
Biomedicals; at 0.833 µg/mL), aggrecan (ACAN; clone 4F4; Santa Cruz; at 0.1
µg/mL), type I collagen (COL1; clone EPR7785; Abcam; at 0.8 µg/mL), type X
collagen (COL10; clone X53 diluted 1:200; generous gift from Prof. Klaus von der
Mark). Antibodies were diluted in 1.25% BSA in PBS, and slides were incubated at
4 °C overnight. Negative controls were made by omitting the primary antibody.
The secondary antibodies, goat anti-mouse IgG conjugated to Alexa 594 and goat
anti-rabbit IgG conjugated to Alexa 488 (both Life Technologies), were diluted
1:400. The stained sections were mounted with ProLong Gold antifade reagent
(Invitrogen), containing DAPI for nuclear staining. Imaging was done using an
upright Nikon Eclipse E600 microscope equipped with an Olympus ColorView III
camera. Thickness of cartilage discs was measured using histological
sections.
Scientific Neg50, Thermo Scientific) and frozen in dry ice-cooled isopentane.
Frozen tissue blocks were stored at −80 °C. The samples were cut in 9- to 10-µm
thick sections on a CryoStar NX70 Cryostat (Thermo Scientific), mounted on
SuperFrost Plus Adhesion slides, stored at −80 °C and fixed for 60 seconds in
cold 95% ethanol directly before starting immunostaining. Sections were
immunostained for the presence of type II collagen (COL2; clone II-4C11; MP
Biomedicals; at 0.833 µg/mL), aggrecan (ACAN; clone 4F4; Santa Cruz; at 0.1
µg/mL), type I collagen (COL1; clone EPR7785; Abcam; at 0.8 µg/mL), type X
collagen (COL10; clone X53 diluted 1:200; generous gift from Prof. Klaus von der
Mark). Antibodies were diluted in 1.25% BSA in PBS, and slides were incubated at
4 °C overnight. Negative controls were made by omitting the primary antibody.
The secondary antibodies, goat anti-mouse IgG conjugated to Alexa 594 and goat
anti-rabbit IgG conjugated to Alexa 488 (both Life Technologies), were diluted
1:400. The stained sections were mounted with ProLong Gold antifade reagent
(Invitrogen), containing DAPI for nuclear staining. Imaging was done using an
upright Nikon Eclipse E600 microscope equipped with an Olympus ColorView III
camera. Thickness of cartilage discs was measured using histological
sections.
6
Immunohistochemical Analysis of Ocular Oxidative Stress
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The eyes were enucleated quickly and immerged in 4% paraformaldehyde in PBS overnight at 4°C. They were incubated successively in 10% and 20% sucrose at 4°C and then embedded in optimal cutting temperature compound (Sakura finetek, Gentaur, Belgium). Frozen sections of 10 ȝm thickness were obtained for immunohistochemistry. The sections were first pre-incubated 2×30 min in blocking buffer 5% BSA and tween-20 0.1% in PBS). They were then processed to overnight incubation at 4 °C in 4-Hydroxynonenal (HNE) (Calbiochem, 1/300) or acrolein (Cosmo bio, Tokyo, Japan, 1/150) antibodies. After two washes of 30 min, the sections were incubated 2 h at room temperature with goat anti-mouse IgG conjugated to alexa 594 (1/500), lectin PNA conjugated to alexa 488 (1/100, Life technologies) and the nuclear marker DAPI (Sigma, 1/1000). The sections were washed 3 times with PBS before ultimately mounted with fluoromount-G (SouthernBiotech). Images have been obtained with a confocal microscope (Olympus FV1000) at 63x objective. Stacks of consecutive images taken at 500 nm intervals were acquired sequentially with lasers: diode 559 nm, argon 488 nm and diode 405 nm and Z projections of serial sections were reconstructed in the software (FV-10-ASW-4.2) coupled to confocal.
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