Embryos were harvested at different points between E8.5 to E10.5. Embryos at E10.5 were fixed in 4% paraformaldehyde overnight at 4 °C. For immunostaining of whole-mount embryos, after paraformaldehyde fixation, the embryos were sequentially dehydrated in methanol and then incubated in the permeabilization buffer (PBlec) (PBS pH6.8, 1% Tween 20, 1 mM CaCl2, 1 mM MgCl2, 0.1 mM MnCl2) for 20 min at room temperature. After permeabilization, the embryos were incubated with primary antibody rat anti-endomucin or rabbit anti-DHX15 (Abcam, Cambridge, UK) (1:20 dilution) in PBlec buffer overnight at 4 °C. To remove residual primary antibody, embryos were washed with PBT (PBS pH 6.8, 0.1% Tween) for 5 × 10 min. Next, the embryos were incubated with secondary antibody Alexa Fluor 488-conjugated goat anti-rat or Alexa Fluor 488-conjugated goat anti-rabbit (Thermo Fisher, Waltham, MA, USA) (1∶500 dilution) in the dark overnight at 4 °C, and then washed with PBT for 3 × 10 min and postfix in 4% paraformaldehyde. Negative controls for endomucin and DHX15 detection were performed by omitting the primary antibodies in the immunofluorescence reactions. Images were acquired using fluorescence stereomicroscope (Leica Microsystems, Heerbrugg, Switzerland) and immunofluorescence microscope (Nikon Eclipse E600, Kanagawa, Japan) systems.
Alexa fluor 488 conjugated goat anti rat
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488-conjugated goat anti-rat is a secondary antibody conjugated with the Alexa Fluor 488 fluorescent dye. It is designed to detect and visualize rat primary antibodies in various immunoassays and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse e600, by Nikon (3 mentions)
Fluorescence stereomicroscope, by Leica (2 mentions)
Alexa fluor 488 conjugated goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Rabbit anti lyve 1, by Abcam (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Rat anti endomucin, by Abcam (1 mentions)
Alexa fluor 488 conjugated goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 conjugated donkey anti goat, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 conjugated goat anti rat, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 conjugated goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (1 mentions)
Axio observer, by Zeiss (1 mentions)
Goat serum, by Jackson ImmunoResearch (1 mentions)
Ti inverted fluorescent microscope, by Nikon (1 mentions)
Sorafenib, by Selleck Chemicals (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Cell Signaling Technology (1 mentions)
Alexa fluor 555 conjugated goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Phospho p38 thr180 tyr182, by Cell Signaling Technology (1 mentions)
Calcein am fluorescent dye, by Merck Group (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
9 protocols using alexa fluor 488 conjugated goat anti rat
1
Embryonic Immunostaining Protocol
2
Immunostaining of Endothelial Markers
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Tissues were fixed in 4% paraformaldehyde, cryoprotected overnight in a 30% sucrose solution and embedded in optimal cutting temperature medium. Next, 2-µm frozen sections were rehydrated, blocked with 5% normal goat serum and incubated with rat anti-endomucin or rabbit anti-Lyve-1 (Abcam, Cambridge, UK) as a primary antibody overnight at 4 °C. The binding sites of the primary antibodies were revealed with Alexa Fluor 488-conjugated goat anti-rat or Alexa Fluor 488-conjugated goat anti-rabbit (Thermo Fisher, Waltham, MA, USA). Tissues for which immunostaining was performed without primary antibodies were used as negative controls. Slides were mounted using Vectashield (Vector Laboratories, Burlingame, CA, USA) and samples were visualized with a fluorescence microscope (Nikon Eclipse E600, Kanagawa, Japan).
3
Whole-Mount Immunostaining of Mouse Embryos
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Embryos were harvested at different points between E8.5 to E10.5. Embryos at E10.5 were fixed in 4% paraformaldehyde overnight at 4ºC. For immunostaining of whole mount embryos, after paraformaldehyde fixation, the embryos were sequentially dehydrated in methanol and then incubated in the permeabilization buffer (PBlec) (PBS pH6.8, 1% Tween 20, 1mM CaCl2, 1mM MgCl2, 0.1 mM MnCl2)
for 20 minutes at room temperature. After permeabilization, the embryos were incubated with primary antibody rat anti-endomucin (Abcam, Cambridge, UK)
(1:20 dilution) in PBlec buffer overnight at 4°C. To remove residual primary antibody, embryos were washed with PBT (PBS pH6.8, 0.1% Tween) for 5×10 minutes. Next, the embryos were incubated with secondary antibody Alexa Fluor 488-conjugated goat anti-rat (Thermo Fisher, Waltham, MA, USA) (1∶500 dilution) in the dark overnight at 4ºC, and then washed with PBT for 3×10 minutes and postfix in 4% paraformaldehyde. Images were acquired using fluorescence stereomicroscope (Leica Microsystems, Heerbrugg, Switzerland) and immunofluorescence microscope (Nikon Eclipse E600, Kanagawa, Japan) systems.
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this this version posted November 3, 2020. ; https://doi.org/10.1101/2020.11.03.366286 doi: bioRxiv preprint
for 20 minutes at room temperature. After permeabilization, the embryos were incubated with primary antibody rat anti-endomucin (Abcam, Cambridge, UK)
(1:20 dilution) in PBlec buffer overnight at 4°C. To remove residual primary antibody, embryos were washed with PBT (PBS pH6.8, 0.1% Tween) for 5×10 minutes. Next, the embryos were incubated with secondary antibody Alexa Fluor 488-conjugated goat anti-rat (Thermo Fisher, Waltham, MA, USA) (1∶500 dilution) in the dark overnight at 4ºC, and then washed with PBT for 3×10 minutes and postfix in 4% paraformaldehyde. Images were acquired using fluorescence stereomicroscope (Leica Microsystems, Heerbrugg, Switzerland) and immunofluorescence microscope (Nikon Eclipse E600, Kanagawa, Japan) systems.
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this this version posted November 3, 2020. ; https://doi.org/10.1101/2020.11.03.366286 doi: bioRxiv preprint
4
Lysosomal Damage Evaluation in α-Synuclein
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For evaluation of lysosomal damage, cells were incubated for 30 min with Lyso Tracker red (dilution, 1:10000) and then were stimulated with α-Syn A53T human or α-Syn human. Alexa Fluor 488–conjugated goat anti-mouse, Alexa Fluor 568–conjugated donkey anti-goat, Alexa Fluor 647–conjugated goat anti-rat and Alexa Fluor 488–conjugated goat anti-rat were purchased from Invitrogen. Lysosomal damage was assessed by Living cell system (Perkin Elmer, USA).
5
Pulse-chase DNA replication kinetics
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Cells were labeled with 50 μM IdU for 30 min and then 50 μM CldU for 30 min. Cells were then trypsinized and resuspended in ice-cold PBS at 2.5 × 105 cell/ml. DNA spreads were made as previously described63 (link) with modifications. Briefly, 2 μl cell suspensions were mixed with 8 μl spreading buffer (200 mM Tris-HCl pH 7.4, 50 mM EDTA, 0.5% SDS) on a silane-coated slide. After 5 min, the slides were tilted at 15° to allow DNA to run down the slide, air dried, and then fixed in methanol/acetic acid (3:1). The slides were treated with 2.5 N HCl at 37 °C for 1 h, washed 3 times in PBS, and blocked with 2% bovine serum albumin in PBS. The slides were then incubated with mouse anti-BrdU/IdU and rat anti-BrdU/CldU in blocking buffer at 4 °C overnight. After washing, the slides were incubated in secondary antibody mixture of Alexa Fluor 488-conjugated goat anti-rat and Alexa Fluor 568-conjugated goat anti-mouse (Invitrogen) at room temperature for 1 h. The slides were then washed and mounted in Prolong gold antifade (Invitrogen). Microscopy was carried out using a Zeiss Axio Observer with oil immersion lens. Fiber length was measured based on a conversion factor of 1 µm to 2.59 kb.
6
Cardiac Fibroblast Immunofluorescence Assay
7
Evaluation of a Novel Anti-Angiogenic Compound
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VH02, 1-((1-(1H-indazol-6-yl)-1H-1,2,3-triazol-4-yl)methyl)-3-(3-chloromethylphenyl)urea was obtained from Kingkarn Sanphanya, The Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mahidol University (Bangkok, Thailand). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, and trypsin-EDTA were all purchased from Gibco BRL (Gaithersburg, MD, USA). VEGF165aa human recombinant protein, monoclonal antibodies against β-actin, VEGFR2, phospho-VEGFR2 (Tyr1175), Akt, phospho-Akt (Ser473), p38 MAPK, phospho-p38 (Thr180/Tyr182), FAK and phospho-FAK (Tyr397), and 4′-6-diamidino-2phenylindole (DAPI) were provided from Cell Signaling Technology (Beverly, MA, USA). Calcein AM fluorescent dye, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT), anti-mouse, and-rabbit IgG-peroxidase antibodies were ordered from Sigma-Aldrich (St. Louis, MO, USA). Matrigel and CD31 mAb were obtained from BD Bioscience (San Jose, CA, USA). Sorafenib was purchased from Selleckchem (Houston, TX, USA). Alexa Fluor 488-conjugated goat anti-rat, Alexa Fluor 555-conjugated goat anti-rabbit, and Topro-3 iodide were obtained from Invitrogen (Eugene, OR, USA). All other chemicals used in this study were analytical grade.
8
Immunofluorescence Imaging of SOX4 and Cytokeratin 8
9
Measuring DNA replication dynamics in HSCs
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DNA molecular combing coupled with FISH was performed as described in Suram et al (2012 (link)) with minor modifications. HSCs were sequentially labeled with 25 μM IdU for 1 h, followed by 200 μM CldU for one additional hour in the culture medium. Since mouse HSCs were maintained in suspension, no medium changes were performed between the two labelings, and a higher concentration of the second analogue was used in order to compete over the first one during incorporation into the replicating DNA. Cells were then harvested and centrifuged twice and incubated for 3 h in media without IdU/CldU. This procedure was repeated three times. Halogenated nucleotides were detected with specific primary antibodies (IdU: mouse anti-BrdU, Becton Dickinson; CldU: rat anti-BrdU, Abcam) and secondary antibodies (Alexa Fluor 647-conjugated goat anti-mouse, Molecular Probes; Alexa Fluor 488-conjugated goat anti-rat; Molecular Probes). Images were acquired with a spinning disk fluorescence microscope, and labeled DNA molecules were individually manually measured by ImageJ software and analyzed by Excel.
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