CMSCs were used for the endothelial cell induction protocol. Cells were seeded at a density of 3,000 cells per cm2 on 24-well plates in EGM-2 culture medium with supplements (Lonza, Walkersville, MD, USA) and cultured for 10 days. The expression of CD31, an endothelial cell marker, was then evaluated by immunofluorescence (BD Biosciences). On the following day, sections were incubated with Alexa fluor 594-conjugated anti-goat IgG (Molecular Probes, Carlsbad, CA, USA). To induce the formation of capillary-like structures, 24-well plates were covered with 250 μL of Matrigel (BD Biosciences) diluted 1:1 in EGM-2. CMSCs were seeded at a density of 30,000 cells per cm2 and cultured for 24 hours. The formation of capillary-like structures was observed over time by using an inverted microscope (Olympus). Additionally, the percentage of CD31 cells before and after EGM-2 induction was evaluated by flow cytometry with APC-conjugated anti-CD31 (BD Biosciences) diluted 1:50 and by analysis with the LSRFortessa flow cytometer.
Alexa fluor 594 conjugated anti goat igg
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan
Alexa Fluor 594-conjugated anti-goat IgG is a secondary antibody conjugated with the Alexa Fluor 594 fluorescent dye. It is designed to detect and visualize goat primary antibodies in various immunodetection techniques, such as immunofluorescence, flow cytometry, and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Axio observer inverted microscope, by Zeiss (2 mentions)
Prolong with dapi, by Thermo Fisher Scientific (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Egm 2 culture medium, by Lonza (1 mentions)
Apc conjugated anti cd31, by BD (1 mentions)
Matrigel, by BD (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Axioplan 2 fluorescent microscope system, by Zeiss (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Vectashield hardset mounting medium with dapi h 1500, by Vector Laboratories (1 mentions)
Troponin t, by Santa Cruz Biotechnology (1 mentions)
Leicacm1850uv cryostat, by Leica (1 mentions)
Anti connexin 43, by Santa Cruz Biotechnology (1 mentions)
Fluoview 1000 confocal microscope, by Olympus (1 mentions)
Cy3 conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated anti goat igg, by Thermo Fisher Scientific (1 mentions)
E400 fluorescence microscope, by Nikon (1 mentions)
10 protocols using alexa fluor 594 conjugated anti goat igg
1
Endothelial Cell Induction and Capillary-like Structure Formation
2
Neutrophil Adhesion Assay for S. sanguinis
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Neutrophils (5x105 cells) were seeded into each well of poly-L-lysine (Sigma Aldrich)-coated 8-well chamber slides (Nalge Nunc, NY, USA) and incubated for 30 min to allow the cells to attach to the well bottom. Following a medium change, cells were infected with the S. sanguinis strains at an MOI of 10. Exogenous addition of H2O2 or 200 nM of PMA served as a positive control. After 3 h of incubation, cells were fixed with PBS containing 4% paraformaldehyde at room temperature for 20 min. Next, cells were blocked overnight with PBS containing 1% bovine serum albumin (Sigma Aldrich) at 4°C and reacted with a goat anti-human elastase polyclonal antibody (1:2000, Santa Cruz Biotechnology, CA, USA) at room temperature for 1 h. After washing with PBS, cells were incubated with Alexa Fluor 594-conjugated anti-goat IgG (1:1000, Molecular Probes, OR, USA) at room temperature for 1 h. Finally, each slide was enclosed with ProLong Gold Antifade Reagent with DAPI (Life Technologies) and observed using a Carl Zeiss Axioplan 2 fluorescent microscope system.
3
Immunofluorescence Imaging of Chagasic Mouse Hearts
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The hearts obtained from chagasic mice were cryopreserved in Tissue-Tek (Sakura, Alphen aan den Rijn, The Netherlands), and 10-μm sections were obtained in Leica CM 1850 UV cryostat (Leica Microsystems, Wetzlar, Germany). To evaluate the presence of GFP+ cells in the heart, sections were incubated overnight with the following primary antibodies: anti-connexin 43 and troponin T (Santa Cruz Biotechnology, Inc.). On the following day, sections were incubated with Alexa fluor 594-conjugated anti-goat IgG or Alexa fluor 488-conjugated anti-rabbit IgG (Molecular Probes, Carlsbad, CA, USA). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) by using VectaShield Hard Set mounting medium with DAPI H-1500 (Vector Laboratories, Burlingame, CA, USA). Sections were then analyzed by using a FluoView 1000 confocal microscope (Olympus).
4
Immunofluorescence Staining of Alopecia Areata
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Paraffin blocks of the biopsied specimens from the hairless patches of 9 patients with AA (type 1, n=3; type 2, n=3; type 3, n=3; and type 4, n=0) were cut into 4-µm thick sections and incubated at 58℃ overnight. The paraffin sections were deparaffinized with 100% xylene two times for 10 minutes each and washed with graded concentrations (100%, 90%, 70%, and 50%) of ethyl alcohol, followed by application of 0.3% PBST solution for 10 minutes, a wash with PBS solution, and then application of 10% blocking solution for 60 minutes. The following primary antibodies were used: mouse monoclonal anti-CCR5 antibody (clone 45549; R&D Systems, Abingdon, UK) for the Th1 fraction, and rabbit polyclonal anti-CCR6 antibody (clone ab78429; Abcam, Cambridge, UK) for the Th17 fraction. These primary antibodies were applied to slide sections at 4℃ and incubated overnight. After slide sections were washed with PBS solution, Cy3-conjugated anti-mouse IgG (Invitrogen, CA, USA), Alexa Fluor 594-conjugated anti-goat IgG (Invitrogen) or Oregon Green 488-conjugated anti-rabbit IgG (Invitrogen) were applied as secondary antibodies at 22℃ for 3 hours. Subsequently, after washing with PBS solution, the slides were observed using confocal microscope (LSM 510; Carl Zeiss, Jena, Germany) and photographed.
5
Immunofluorescence Staining of Frozen Sections
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Frozen sections were probed with primary antibody in PBS/ 0.01% Triton X-100 at 4 °C overnight. All primary antibodies were listed in Supplementary Table 2. The sections were then incubated with Alexa Fluor 488-conjugated anti-goat IgG or Alexa Fluor 594-conjugated anti-goat IgG (Invitrogen) secondary antibodies for 2 h. Images were acquired on a Nikon E400 fluorescence microscope (Tokyo, Japan). Digitally captured images were analyzed using NIS-Elements imaging software (Nikon, Tokyo, Japan).
6
Visualizing Mitochondrial Morphology in Cells Expressing DJ-1 Variants
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Cells transiently transfected with wt and mutant DJ-1 were cultured on poly-D -lysine glass coverslips (24Ø) for 24 h. After administration of CCCP 10 μM for 8 h, cells were washed twice in cold PBS (pH 7.4) and fixed in 4% (w/v) paraformaldehyde for 20 min at RT. Following three washes in PBS, cells were permeabilized with 0.1% Triton-X100 (AppliChem PanReac) for 10 min at RT and blocked with 3% (w/v) bovine serum albumin for 1 h at RT. All coverslips were then incubated with primary antibodies (goat polyclonal anti-DJBP at 0.4 μg/ml, Santa Cruz) and rabbit polyclonal anti-ATPβ at 1 μg/ml (Abcam) at 4°C overnight. Following three washes in PBS, coverslips were incubated in the dark with a mix of the following secondary antibodies AlexaFluor 594-conjugated anti-goat IgG and AlexaFluor 488-conjugated anti-rabbit IgG (Invitrogen) at 1:100 for 1 h at RT. After a final wash, coverslips were mounted with ProLong Gold antifade reagent (Invitrogen) containing DAPI (Applichem), for nuclei visualization, and imaged by means of an Olympus Fluoview 1000 Confocal Laser Scanning System. All images were analyzed by ImageJ software (NIH, Bethesda, MD, United States).
7
Immunofluorescent Staining of hACE2
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To stain for surface-expressed hACE2, BHK21-hACE2 or the control BHK21 cells seeded onto fibronectin-coated glass coverslips were incubated with 0.4 μg/mL of hACE2-specific goat antibody (#AF933, R&D systems) at 4°C in media containing 25 mM HEPES. Next, cells were washed with cold PBS, fixed with 4% paraformaldehyde, and blocked with buffer (2% (w/v) bovine serum albumin, 5% (v/v) glycerol, 0.2% (v/v) Tween20 in Ca2+/Mg2+-free PBS). Secondary donkey AlexaFluor 594-conjugated anti-goat IgG (#A32758 Invitrogen) was used to detect the hACE2 signal. Coverslips were mounted in Prolong with DAPI (Invitrogen) and imaged on an Axio Observer inverted microscope (Zeiss).
8
Neuronal and Glial Activation in Mouse Brain
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Mice were placed into Context A and explored for 5 min freely. Then we put the mice back to their home cages. Following a 1.5-h break, mice were anesthetized with isoflurane and transcardially perfused with 0.01 M phosphate-buffered saline (PBS), pH 7.4 followed by 4% paraformaldehyde in 0.2 M PBS, pH 7.4. Brains were rapidly removed and fixed using the same fixative for 4 h at 4 °C. After dehydrating twice with 30% sucrose for 48 h at 4 °C, brain sections (50 μm) were sliced on a freezing microtome. The sections from each group (3–6 sections from each region) were rinsed in a blocking solution for 2 h and incubated overnight (4 °C) with primary antibodies: c-Fos (1:1500, CST), glial fibrillary acidic protein (GFAP) (1:250, Gene Tex), ionized calcium-binding adaptor molecule 1 (IBA1) (1:500, WAKO). Sections were then washed with PBS and probed with secondary antibodies: Alexa Fluor 594-conjugated anti-rabbit secondary IgG (Invitrogen) or Alexa Fluor 594-conjugated anti-goat IgG (Invitrogen) for 4 h. All images were captured with a confocal microscope (FV3000, Olympus, Japan), and processed with the Imaris software (v.7.7.1, Bitplane, Switzerland). For cell counting, 3–6 sections per region were counted per animal with the Imaris software by experimenters blinded to the groups.
9
Immunofluorescent Detection of hACE2
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To stain for surface-expressed hACE2, BHK21, A549, BHK21-hACE2 and A549-hACE2 cells or the control cells were seeded onto fibronectin-coated glass coverslips were incubated with 0.4 μg/mL of hACE2-specific goat antibody (#AF933, R&D systems) at 4°C in media containing 25 mM HEPES. Next, cells were washed with cold PBS, fixed with 4% paraformaldehyde, and blocked with buffer (2% (w/v) bovine serum albumin, 5% (v/v) glycerol, 0.2% (v/v) Tween20 in Ca2+/Mg2+-free PBS). Secondary donkey AlexaFluor 594-conjugated anti-goat IgG (#A32758 Invitrogen) was used to detect the hACE2 signal. Coverslips were mounted in Prolong with DAPI (Invitrogen) and imaged on an Axio Observer inverted microscope (Zeiss).
10
Immunofluorescence Staining of Mouse Brain
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Following treatment, the mice were anaesthetised and transcardially perfused with ice-cold PBS and fixed by the perfusion with 4% paraformaldehyde in PBS. Brain tissues were harvested and the harvested tissues were fixed in 4% paraformaldehyde again for 24 h followed by incubation in 80% ethanol. A coronal section (1 mm thick) was cut using a mouse brain slicer (Muromachi Kikai, Tokyo, Japan). Each section was embedded in paraffin. For immunofluorescence staining, 1-3 µm thick sections were de-paraffinized with xylene and rehydrated with 80% ethanol. Staining was performed using goat anti-Iba1 (Wako, Osaka, Japan), Alexa Fluor 488 conjugated rabbit anti-CD68 (Bioss Antibodies, Woburn, MA), rabbit anti-TMEM119 (Cell Signaling Technology, Danvers, MA), rat anti-BrdU (Abcam, Eugene, OR), rabbit anti-NeuN (Merck Millipore, Darmstadt, Germany), rabbit anti-GFAP (Bioss Antibodies), and rabbit anti-cleaved caspase3 (Cell Signaling Technology) antibodies. Next, the sections were incubated with Alexa Fluor 594 conjugated anti-goat IgG (Invitrogen, Waltham, MA), Alexa Fluor 594 conjugated anti-rabbit IgG (Invitrogen), or Alexa Fluor 488 conjugated anti-rat IgG (Invitrogen). Computer-assisted morphometric analysis was performed using a digital microscope controller (BZ-8000, Keyence Co., Osaka, Japan) and ImageJ software (NIH, Bethesda, MD, USA) was used for image analysis.
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