Hematoxylin & Eosin staining was performed as described (Wang et al., 2008 (link)). For flat mount staining of the choroidal vasculature, pigment bleaching in RPE cells and choroidal melanocytes was performed as described (Kim and Assawachananont, 2016 ). Briefly, pigmented eyes were fixed in 4% paraformaldehyde PBS for 30 min to 2 h. After washing with PBS three times, the cornea and lens were removed before 3% hydrogen peroxide bleaching at 55 °C for 30 min to 2 h depending on the stage, or at 4 °C for several days. Immunofluorescence staining was performed using standard procedures. Primary antibodies used include: Beta-galactosidase (LacZ) from chicken (1:300, Abcam), Endomucin from rat (1:200, Santa Cruz Biotechnology), PECAM-1 from rat (1:50, BD Pharmingen), ICAM-2 from rat (1: 200, BD Pharmingen). These three EC markers, Endomucin, PECAM-1 and ICAM-2, were used interchangeably, but some antibodies worked better than others due to different sample processing. Secondary antibodies include: goat antichicken (or rat) AlexaFluor 488 (1:500, Invitrogen) and goat anti-rat AlexaFluor 594 (1:500, Invitrogen).
Goat anti rat alexafluor 594
Manufactured by Thermo Fisher Scientific
Goat anti-rat AlexaFluor 594 is a secondary antibody conjugated with the fluorescent dye AlexaFluor 594. It is designed to detect and visualize the presence of rat-specific primary antibodies in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (4 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Beta galactosidase lacz from chicken, by Abcam (2 mentions)
Endomucin from rat, by Santa Cruz Biotechnology (2 mentions)
Pecam 1 from rat, by BD (2 mentions)
Icam 2 from rat, by BD (2 mentions)
Bx61 fluorescence microscope, by Olympus (2 mentions)
Prolong diamond antifade mounting medium, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Eclipse 80i fluorescence microscope, by Nikon (1 mentions)
Bu1 75 icr1, by Abcam (1 mentions)
Ab6326, by Abcam (1 mentions)
Mouse anti brdu clone b44, by BD (1 mentions)
Mouse anti brdu, by BD (1 mentions)
Rat anti cldu, by Novus Biologicals (1 mentions)
Anti ki67, by Abcam (1 mentions)
Anti lyve 1, by Abcam (1 mentions)
Anti prox1, by Abcam (1 mentions)
Anti pecam1, by BD (1 mentions)
10 protocols using goat anti rat alexafluor 594
1
Choroidal Vasculature Flat Mount Staining
2
DNA Fiber Analysis of CldU and IdU Incorporation
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Cells were pulse-labelled with 50 μM 5-chloro-2′-deoxyuridine (CldU) and 250 μM 5-iodo-2′-deoxyuridine (IdU) as indicated, with or without treatment as reported in the experimental schemes. DNA fibers were spread out as previously reported (26 (link)). For immunodetection of labelled tracks the following primary antibodies were used: anti-CldU (1:60; rat-monoclonal anti-BrdU/CldU; BU1/75 ICR1 Abcam) and anti-IdU (1:10; mouse-monoclonal anti-BrdU/IdU; clone b44 Becton Dickinson). The secondary antibodies were goat anti-mouse Alexa Fluor 488 or goat anti-rat Alexa Fluor594 (Invitrogen). The incubation with antibodies was accomplished in a humidified chamber for 1 h at 37°C. Images were acquired randomly from fields with untangled fibers using Eclipse 80i Nikon Fluorescence Microscope, equipped with a Video Confocal (ViCo) system. The length of labelled tracks was measured using the Image-Pro-Plus 6.0 software. A minimum of 100 individual fibers were analyzed for each experiment and the mean of at least three independent experiments presented. Statistics were calculated using GraphPad Prism Software.
3
DNA Fiber Analysis for Replication Dynamics
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DNA fiber analysis was performed essentially as in ref. 47 . Briefly, ongoing DNA synthesis was labeled with the indicated nucleotide analogs, cells were washed with PBS, lysed with lysis buffer (0.5% SDS, 200 mM Tris–HCL (pH 7.4) and 50 mM EDTA) and spread on glass slides by tilting. After drying, the slides were fixed in methanol: acetic acid (3:1), dried, and rehydrated in PBS. DNA was denatured by incubating the slide in 2.5 N hydrochloric acid for 1 h. After neutralization in PBS, samples were blocked in blocking buffer (10% NGS in PBS), and stained with primary antibodies (Mouse Anti-BrdU Clone B44 (BD, #347580) for IdU, Abcam ab6326 for CldU, both 1:50 in the blocking buffer), washed with PBS-1%Tween20, incubated with fluorescently labeled secondary antibodies (Goat anti-mouse AlexaFluor 488 (Invitrogen, #A-11001), Goat anti-rat AlexaFluor 594 (Invitrogen, #A-11007), both 1:150 in blocking buffer). After extensive washes with PBS-Tween20 and PBS, slides were mounted with Prolong Diamond Antifade mounting medium (Invitrogen, #P36961). Samples were imaged using Olympus BX61 fluorescence microscope at 60x magnification; image analysis was performed using Fiji (ImageJ) software. At least 100 tracks per sample were analyzed.
4
Detecting Single-Stranded DNA in Cells
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In order to assess the presence of ssDNA in the cells, cells were treated with 20 µM CldU for 48 h, dox/aux were added to the indicated samples for the last 16 h of treatment. After two PBS washes, cells were briefly extracted with CSK buffer (10 mM PIPES pH 7.0, 300 mM Sucrose, 100 mM NaCl, 3 mM MgCl2, 0.5% Triton X-100; 5 min on ice), followed by fixation with 4% paraformaldehyde for 10 min at room temperature. After washes with PBS, samples were blocked with 5% BSA and stained with anti-BrdU antibody (ab6326, 1:150) followed by the incubation with secondary antibody, goat anti-rat AlexaFluor 594 (Invitrogen #A-11007, 1:300). After extensive PBS washes, nuclei were stained with 0.1 µg/ml Hoechst 33342 for 10 min and after a brief wash with PBS, samples were mounted using Prolong Diamond Antifade mounting medium (Invitrogen, #P36961). Samples were imaged using Olympus BX61 fluorescence microscope at 60x magnification; cell containing over 5 bright ssDNA foci were considered ssDNA-positive.
5
Dual-Pulse Labeling for DNA Replication Analysis
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Unsynchronized cells were pulse-labeled for 30 min by a medium containing 100 μM of the thymidine analog iododeoxyuridine (IdU). At the end of the first labeling period, the cells were washed twice with a warm medium and pulse-labeled once more for 30 min with a medium containing 100 μM of another thymidine analog chlorodeoxyuridine (CldU). Cells were then harvested, and genomic DNA was extracted, combed, and analyzed as previously described (Lebofsky et al, 2006 (link); Herrick & Bensimon, 2009 (link)). The primary antibody for fluorescence detection of IdU was mouse anti-BrdU (Becton Dickinson), and the secondary antibody was goat anti-mouse Alexa Fluor 488 (Invitrogen). The primary antibody for fluorescence detection of CldU was rat anti-CldU (Novus Biologicals). The secondary antibody was goat anti-rat Alexa Fluor 594 (Invitrogen). The length of the replication signals and the distances between origins were measured in micrometers and converted to kilo bases according to a constant and sequence-independent stretching factor (1 μm = 2 Kb), as previously reported (Herrick & Bensimon, 2009 (link)).
6
Embryonic Skin Lymphatic Development
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Mouse embryos were fixed in 4% paraformaldehyde overnight at 4°C. Dorsal skin from mouse embryos was dissected and labeled as described [41 (link)] (S1 Fig ). Embryonic skin was labeled with anti-PROX1 (1:2000, Abcam), anti-LYVE1 (1:100, Abcam), anti-PODOPLANIN (1:100, Angiobio), anti-Ki67 (1:100, Abcam) and anti-PECAM1 (1:100, BD biosciences). Secondary antibodies used were goat anti-hamster Alexa fluor 488 (1:100), goat anti-rabbit Alexa fluor 488 (1:100), goat anti-rabbit Alexa fluor 594 (1:100) and goat anti-rat Alexa fluor 594 (1:100) from Invitrogen.
7
Choroidal Vasculature Flat Mount Staining
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Hematoxylin & Eosin staining was performed as described (Wang et al., 2008 (link)). For flat mount staining of the choroidal vasculature, pigment bleaching in RPE cells and choroidal melanocytes was performed as described (Kim and Assawachananont, 2016 ). Briefly, pigmented eyes were fixed in 4% paraformaldehyde PBS for 30 min to 2 h. After washing with PBS three times, the cornea and lens were removed before 3% hydrogen peroxide bleaching at 55 °C for 30 min to 2 h depending on the stage, or at 4 °C for several days. Immunofluorescence staining was performed using standard procedures. Primary antibodies used include: Beta-galactosidase (LacZ) from chicken (1:300, Abcam), Endomucin from rat (1:200, Santa Cruz Biotechnology), PECAM-1 from rat (1:50, BD Pharmingen), ICAM-2 from rat (1: 200, BD Pharmingen). These three EC markers, Endomucin, PECAM-1 and ICAM-2, were used interchangeably, but some antibodies worked better than others due to different sample processing. Secondary antibodies include: goat antichicken (or rat) AlexaFluor 488 (1:500, Invitrogen) and goat anti-rat AlexaFluor 594 (1:500, Invitrogen).
8
Immunofluorescent Staining of Cells and Tissues
9
Immunofluorescence Analysis of Ovarian Markers
10
Immunohistochemistry of α-Synuclein, CD11b, and Myosin IIB
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Cultures were fixed in 4% paraformaldehyde (PFA, Sigma-Aldrich) dissolved in PBS (Biochrom GmbH, Berlin, Germany) for 15 min and permeabilized by washing them three times for 5 min with PBS containing 0.1% Triton X-100 (PTX). Blocking solution containing PTX and 5% normal goat serum (Vector Laboratories, Burlingame, CA, USA) was applied for 30 min. The primary antibodies mouse anti-α-synuclein (1:500; BioLegend), rat anti-CD11b (1:250; Serotec by Bio-Rad), or mouse anti-non-muscle Myosin IIB (1:500; abcam) were applied for 1 h followed by three washing steps. The secondary antibodies goat anti-mouse-AlexaFluor594 (1:250; Invitrogen) and goat anti-rat-AlexaFluor594 (1:250; Invitrogen) were applied for 30 min. Texas Red-X Phalloidin (1:1,000, Thermo Fisher Scientifics) was applied for 20 min without prior blocking steps. 4’,6-Diamidino-2’-phenylindol-dihydrochloride (DAPI, Sigma-Aldrich) was used for nuclear counterstaining at 0.1 mg/mL for 20 min in PBS. Images were taken using a 60x oil-objective.
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