Immunohistochemistry was performed according to standard protocols, and the following primary antibodies were used: pERK (rabbit, 1: 200, Cell Signaling, Danvers, MA, USA), Alexa Fluor® 488 (donkey anti-rabbit IgG, 1:1000, Molecular Probes, Waltham, MA), CGRP antibody (goat, 1:300, Abcam, Cambridge, MA), CCR2 antibody (rabbit, 1:300, Novus Biologicals, Centennial, CO, USA), Alexa Fluor® 594 (donkey anti-rabbit IgG, 1:1000, Abcam), pERK antibody (mouse, 1: 300, Abcam), and Alexa Fluor® 594 (donkey anti-mouse IgG, 1:1000, Abcam).
Alexa fluor 594
Manufactured by Abcam
Sourced in United Kingdom, United States, Canada, Germany, France, China
Alexa Fluor 594 is a fluorescent dye that can be used in various biomedical applications, such as immunohistochemistry and flow cytometry. It has an excitation maximum at 590 nm and an emission maximum at 617 nm, making it suitable for detection in the red spectrum.
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Lab products found in correlation
Alexa fluor 488, by Abcam (51 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (11 mentions)
Alexa fluor 647, by Abcam (8 mentions)
Triton x 100, by Merck Group (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions)
Bovine serum albumin (bsa), by Merck Group (5 mentions)
Alexa fluor 555, by Abcam (5 mentions)
Ab150080, by Abcam (4 mentions)
Fv10i, by Olympus (4 mentions)
Lsm 780, by Zeiss (4 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (3 mentions)
Ab150077, by Abcam (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (3 mentions)
Fluorescence microscope, by Leica (3 mentions)
Goat anti rabbit igg h l, by Abcam (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (3 mentions)
Ab104139, by Abcam (3 mentions)
α sma, by Abcam (2 mentions)
Triton x 100, by Beyotime (2 mentions)
166 protocols using alexa fluor 594
1
Immunohistochemical Analysis of Signaling Pathways
2
Visualizing MCHR1 membrane localization
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HEK293T cells were seeded on poly-L-lysine pretreated coverslips of 12-well plates and transfected with MCHR1-F1 and MRAP1-Flag-F2 or MRAP2-Flag-F2. Each well was transfected with 1 μg of plasmids in total. The next day, cells were fixed with 4% PFA Fix Solution for 20 min. Cells were incubated with anti-FLAG antibody (Cell Signaling) at 1:5,000 for 2 h at room temperature for the detection of MRAP1 and MRAP2 proteins. Then, samples were washed 3 times and incubated with 1:5,000 secondary antibody Alexa Fluor594 (Abcam) for 2 h at room temperature. To detect membrane translocation of MCHR1 in the presence of MRAP2, we transfected GFP-MCHR1 and MRAP2 or RAMP3 at a ratio of 1: 9 without antibody treatment. In order to observe co-fluorescence of MCHR1 and MRAP2 chimeras, 3HA-MCHR1 and each 2xFLAG MRAP2 chimera were transiently transfected. Cells were incubated with both anti-HA and anti-FLAG antibody (Cell Signaling) at 1:5,000 for 2 h at room temperature. Samples were then washed 3 times and incubated with both 1:5,000 secondary antibody Alexa Fluor488 (Abcam) and Alexa Fluor594 (Abcam) for 2 h at room temperature.
Coverslips were fixed with nail polish on the glass slide containing ProLong (R) Gold Antifade with DAPI Molecular Probes (Cell Signaling). Imaging was collected using a 63× oil objective with laser-scanning Zeiss confocal microscopy (LSM880).
Coverslips were fixed with nail polish on the glass slide containing ProLong (R) Gold Antifade with DAPI Molecular Probes (Cell Signaling). Imaging was collected using a 63× oil objective with laser-scanning Zeiss confocal microscopy (LSM880).
3
Immunofluorescent Staining of Injured Mice
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The preparation of tissue slices, immunofluorescence experiments and analysis were carried out based on our previous methods (Zhao et al., 2022 (link)). Mice were euthanized at 2 weeks post injury (2 WPI). The primary antibodies were as follows: glial fibrillary acidic protein (GFAP, Cat# ab4674, Abcam, 1:500), S100 family protein p11 (S100A10, Cat# 11250-1-AP, Protein Tech, 1:250), and complement C3 (C3, Cat# sc-528410, Santa Cruz Biotechnology, 1:50); the secondary antibodies were Alexa® Fluor 488 (Cat# abs20019A, Absin, 1:500), Alexa® Fluor 488 (Cat# A-32814, Invitrogen, 1:500), Alexa® Fluor 555 (Cat# A-32773, Invitrogen, 1:500), Alexa Fluor® 594 (Cat# ab150156, Abcam, 1:500), Alexa® Fluor 594 (Cat# ab150176, Abcam, 1:500), and Alexa® Fluor 647 (Cat# A-32795, Invitrogen, 1:500). Fluorescent antibodies of the same channel including Alexa® Fluor 488 and Alexa® Fluor 594, were used to match species differences during immunofluorescence staining.
4
Immunocytochemical Analysis of Primary LEC
5
Vimentin and α-SMA Immunostaining
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CAFs and NFs were probed with primary antibodies against vimentin (Abcam) and α-SMA (Abcam) overnight at 4 °C. Next, cells were incubated with secondary antibody conjugated with fluorescent Alexa Fluor® 594 (Abcam) in darkness. Finally, cells were captured with a fluorescence microscope. Nuclei were stained with DAPI for 15 min.
6
Astrocyte and Neuron Characterization
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The isolated astrocytes or neurons were characterized by staining marker GFAP or Neun. Firstly, cells were put in 4% paraformaldehyde (Biosharp, Beijing, China) for 20 min. After that, put them in PBS containing 0.1% Triton X100 (Beyotime, Shanghai, China) for 20 min. Then incubated them with 5 % BSA (Biofroxx, Shanghai, China) for 1 h. After above, primary astrocytes were incubated with rabbit monoclonal antibody anti-GFAP (1:200; #80788S; CST, USA) and primary neurons were incubated with a rabbit monoclonal antibody anti-NeuN (1:200; #24307; CST) overnight at 4° C and then incubated the next day with goat anti-rabbit IgG H&L (Alexa Fluor® 488) (1:200; ab150077; Abcam, Cambridge, MA, USA) or goat anti-rabbit IgG H&L (Alexa Fluor® 594) (1:200; ab150080; Abcam) and incubated with DAPI (1:300, Cat. # C1002, Beyotime) to stain nuclei. Finally, the images were observed and photographed. The number of GFAP-positive cells or Neun-positive cells was calculated from randomly selected microscopic fields. Astrocyte or neuronal purity was presented as percentage of the number of the percentage of GFAP-positive or Neun-positive cells to the total cells of view, both around 90% or more.
7
Dual Fluorescent Immunostaining of ACE2 and SARS-CoV-2 N
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The sections were labeled with Rb mAb against human ACE2 (Abcam, ab108209) and the SARS-CoV-2 nucleocapsid antibody chimeric Mab (Sino Biological, Inc., Beijing, China. Cat: 40143-MM05TA) at a 1:500 dilution overnight at 4 °C. Finally, human ACE2 protein antigens were visualized via donkey anti-rabbit IgG H&L (Alexa Fluor® 594) (Abcam, ab150076), and SARS-CoV-2 N protein antigens were visualized via goat anti-human IgG Fc (DyLight® 488) (Abcam, ab97003) at a 1:500 dilution for one hour. The images were captured via a Leica TCS SP8 laser confocal microscope.
8
Apoptosis Induction in PSMA-Targeted Tumor Therapy
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Animals bearing PC3pip tumors were divided into 7 groups: (1) mice receiving PBS; (2) mice receiving 100 nmol/kg PSMA-1-MMAE-IR700 with PDT; (3) mice receiving equal doses of PSMA-1-MMAE-IR700 to group 2, but not receiving PDT; (4) Mice receiving equal doses of PSMA-1-IR700 to group 2 with PDT treatment; (5) Mice receiving equal doses of PSMA-1-IR700 to group 2 without PDT; (6) mice receiving equal dose of PSMA-1-IR700 to group 2 + free MMAE with PDT (MMAE normalized to that delivered by PSMA-1-MMAE-IR700); and (7) mice receiving equal dose of PSMA-1-IR700 to group 2 + free MMAE without PDT. Animals were treated with one single dose and were sacrificed at 4-day post treatment. Tumors were snap-frozen in OCT, cut into 10 μm thick sections and fixed on slides. Induction of apoptosis by the treatment was determined by rabbit polyclonal anti-Caspase-3 antibody (Abcam, Cambridge, UK). A goat anti-rabbit polyclonal antibody labeled by Alexa Fluor-594 was used as secondary antibody (Abcam, Cambridge, UK). The presence of apoptosis was determined by fluorescence images under Leica DM4000B fluorescence microscope at 10 ×. H&E staining of tumor tissues was performed in adjacent sections to check the histology of the tumors. Experiments were repeated in 5 mice.
9
Immunofluorescence Staining for Hypoxia Markers
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Eyes were fixed in 4% PFA for 2 h at room temperature and equilibrated in 30% sucrose at 4 °C, followed by embedding in OCT. Sections (10-μm thick) were heated at 98 °C for 10 min in citric acid buffer for antigen retrieval, blocked with 10% goat serum for 1 hour, and incubated with mouse HIF-1α (1:100, BD Biosciences, 610958), rabbit Ki-67 (1:200, RM-9106, Thermo Scientific), rabbit PFKFB3 (1:100, Proteintech, 13763-1-AP), rat CD31 (1:25, Invitrogen, DIA-310) and/ or Alexa-594 labeled Griffonia simplicifolia isolectin B4 (1:100, Invitrogen, Cat. No. 121413) overnight at 4 °C, followed by incubation with fluorescence-conjugated secondary antibody (1:250, Molecular Probes, Life Technologies, Carlsbad, CA,USA) for 1 hour. For Ki-67/ ERG double immunofluorescent staining, sections were then stained with Anti-ERG antibody (Alexa Fluor® 594) (1:200, Abcam, Clone number: EPR3864) overnight at 4 °C. Sections were washed with PBS, immersed in ProLong Gold mounting medium with DAPI (Invitrogen) to visualize the nuclei, and examined using confocal microscopy. For all immunofluorescence experiments, parallel groups of sections were stained with only primary or secondary antibody as negative controls.
10
Phagocytosis Assay in Murine Macrophages
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Macrophages derived from ACE10 mice were seeded into each chamber of 8-chamber culture slides (BD bioscience) in culture medium. Phagocytosis was performed by adding 1μm latex beads (Sigma,1:500 dilution) into the cells and co-incubating for 1 hour at 37 °C. After extensive washing with PBS, cells were fixed with 4% PFA/PBS for 10 min followed by blocking and permeabilization with 1% BSA and 0.1% Triton X-100 in PBS for 1 hour at room temperature (RT). Then, primary antibodies were used for staining at RT for 2 hours followed by treating the cells with secondary antibodies for another 2 hours at RT. After PBS washing, chamber walls were removed and coverslips were mounted with DAPI- mounting medium (Vector). Images were acquired using a laser scanning confocal microscope (Olympus FV10i). A secondary anti-rabbit antibody used to stain rabbit-anti-ACE IgG was Alexa fluor 488-conjugated (Abcam), while other secondary antibodies were all anti-mouse IgG conjugated with Alexa fluor 594 (Abcam).
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