To detect ROPs on PVM via semipermeabilization, confluent monolayers of HFFs were seeded onto coverslips and infected with ROP54HAII parasites at the indicated time points. The samples were washed quickly with PBS and fixed in 4% formaldehyde (Polysciences) for 10 min at room temperature. The fixed coverslips were quenched with 100 mM glycine–PBS for 5 min at room temperature. The cells were permeabilized with either 0.002% digitonin–PBS (made fresh for each experiment) for 2.5 min at 4°C or 0.01% saponin–PBS for 30 min at room temperature. The samples were placed in blocking buffer (10% fetal calf serum [FCS]–PBS) for 30 min at room temperature to prevent nonspecific binding of the antibodies. Primary antibodies were diluted in blocking buffer (1:300 for MAb HA.11 [Covance], 1:300 for pAb ROP5 [Sibley], 1:100,000 for mouse SAG1 [DG52], and 1:100,000 for rabbit pAb SAG1) and used to probe the coverslips at room temperature for 1 h. The secondary antibodies Alexa 488-conjugated goat anti-mouse and Alexa 594-conjugated goat anti-rabbit (Invitrogen) were diluted at 1:2,000 in blocking buffer and added to the samples for incubation for 1 h (27 (link)). The coverslips were mounted in Vectashield (Vector Labs.) or ProLong Gold (Molecular Probes) and viewed with an Axio Imager.Z1 fluorescence microscope (Zeiss).
Alexa 594 conjugated goat anti rabbit
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa-594 conjugated goat anti-rabbit is a secondary antibody that binds to rabbit primary antibodies. It is labeled with the Alexa Fluor 594 fluorescent dye, which can be detected using appropriate filter sets.
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Lab products found in correlation
Alexa 488 conjugated goat anti mouse, by Thermo Fisher Scientific (7 mentions)
Lsm 510, by Zeiss (4 mentions)
Alamarblue cell viability reagent, by Thermo Fisher Scientific (2 mentions)
Flag ha tag, by Addgene (2 mentions)
H3k27me3, by Cell Signaling Technology (2 mentions)
Prolong gold with dapi, by Thermo Fisher Scientific (2 mentions)
Prolong gold, by Thermo Fisher Scientific (2 mentions)
Axio imager z1, by Zeiss (2 mentions)
Chamber slide, by BD (2 mentions)
Formaldehyde, by Polysciences (1 mentions)
Axio imager z1 fluorescence microscope, by Zeiss (1 mentions)
Ha 11, by Fortrea (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Fitc conjugated lectin, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Eclipse ti epifluorescence microscope, by Nikon (1 mentions)
Illustrator cc 2018, by Adobe (1 mentions)
Hoechst, by Santa Cruz Biotechnology (1 mentions)
Prolong diamond, by Thermo Fisher Scientific (1 mentions)
Ab23894, by Abcam (1 mentions)
14 protocols using alexa 594 conjugated goat anti rabbit
1
Detecting ROP54HA Proteins in Parasites
2
Immunofluorescence Imaging of Choroidal Flat-Mounts
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The choroidal flat-mounts were prepared as described previously58 (link). In brief, the eyes of mice that received an intravitreal injection of control vesicles, LMPs, or DiI-LMPs (n ≥ 3 for each group) were enucleated and fixed in 4% PFA. After the cornea, lens and retina was removed, the choroid-sclera was permeabilized in 1.0% Triton X100 and blocked in 10% normal goat serum. A first antibody against the ionized calcium-binding adapter molecule 1 (Iba1) (1:500; Wako Chemicals, USA) and second antibodies the Alexa-448 -conjugated goat anti-rabbit or Alexa-594 conjugated goat anti-rabbit (1:1000; Invitrogen) were used to specifically identify macrophages. FITC-conjugated lectin was used to stain endothelial cells (1:100; Sigma-Aldrich, St. Louis, MO); Rhodamine Phalloidin (R415; 1:400; Santa Cruz Biotechnology, Santa Cruz, CA) was used for RPE cells. Antibody against mouse IL-10-APC (1:1000) and against mouse IL-12-PE clone (1:1000) were used to detect IL-10 and IL-12, respectively. Cell nuclei were stained with DAPI (1:5000; Invitrogen). Labeled flat-mounts were examined with a laser scanning confocal microscope (Zeiss LSM 510).
3
Establishment of MPNST Cell Lines
4
Immunolabeling of Intracellular Parasites
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Intracellular parasites were fixed with 4% formaldehyde at 4°C for 15 min and permeabilized with 0.25% Triton X-100 in PBS for 8 min. After blocking for 10 min with a PBS solution containing 5% normal goat serum (NGS) and 5% heat-inactivated fetal bovine serum (IFS), staining was performed with mouse anti-Ty (clone BB2) (Bastin et al., 1996 (link)), rabbit anti-ALD (a gift from L. David Sibley), or rabbit anti-HSP70 (a gift from Dominique Soldati-Favre) (Pino et al., 2007 (link)). Alexa-488-conjugated goat-anti-mouse (Invitrogen, cat. no. A11029) and Alexa-594-conjugated goat-anti-rabbit (Invitrogen, cat. no. A11037) were used as secondary antibodies. Nuclei were stained with Hoechst (Santa Cruz, cat. no. sc-394039), and coverslips were mounted in Prolong Diamond (Thermo Fisher, cat. no. P36965). Images were acquired using an Eclipse Ti epifluorescence microscope (Nikon) using the NIS elements imaging software. FIJI was used for image analysis, and Adobe Photoshop and Illustrator CC 2018 were used for image processing.
5
Establishment of MPNST Cell Lines
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MPNST724 and ST88-14 human MPNST cell lines were obtained from Jonathan A. Fletcher laboratory at DFCI and have been tested mycoplasma free. MPNST 724 was grown in RPMI with 10% FBS and ST88-14 was grown in RPMI with 15% FBS. cDNA for wild-type human EED and SUZ12 in pDONR vectors were obtained from Harvard PlasmidID and cloned into MSCV-based retroviral vector with FLAG-HA (FH) tag (Addgene plasmid 41033)43 (link) using Gateway technology. To generate stably expressing cell lines, MPNST724 and ST88-14 were infected with empty vector, MSCV-FH-EED, MSCV-FH-SUZ12 and selected using puromycin (2 μg/ml for 72 hours). Growth curve of the infected cells was performed using Alamar blue cell viability reagent (Life Technology, DAL1100).
For immunofluorescence of infected cell lines, cells were fixed in 4% paraformaldehyde for 10 minutes, permeabilized in 0.1% Triton X-100, and blocked for 1 hours using 10% goat serum. The cells were then incubated for 2 hours in primary antibody (H3K27me3, #9733, Cell Signaling, 1:400) followed by secondary antibody (Alexa-594 conjugated goat-anti-rabbit, Invitrogen). Slides were mounted using Prolong Gold with DAPI (Invitrogen). Photographs were taken on a Nikon microscopy using a Roeper Scientific camera.
For immunofluorescence of infected cell lines, cells were fixed in 4% paraformaldehyde for 10 minutes, permeabilized in 0.1% Triton X-100, and blocked for 1 hours using 10% goat serum. The cells were then incubated for 2 hours in primary antibody (H3K27me3, #9733, Cell Signaling, 1:400) followed by secondary antibody (Alexa-594 conjugated goat-anti-rabbit, Invitrogen). Slides were mounted using Prolong Gold with DAPI (Invitrogen). Photographs were taken on a Nikon microscopy using a Roeper Scientific camera.
6
Limbal Stem Cell Immunophenotyping
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Limbal biopsies from human corneas (n = 3) were isolated under a dissecting microscope and small pieces of tissue were embedded in OCT compound. Thick frozen sections (7 μm) were cut from the crypt rich and non-crypt rich limbus tangential to the corneal circumference on a cryostat and mounted on superfrost plus microscope slides (VWR International, West Sussex, UK). Frozen sections were fixed with 4% PFA, permeabilized with 0.5% Triton-X and blocked with 10% goat serum in PBS at room temperature for 90 minutes. Sections were then incubated with primary antibodies against CD90 (Abcam ab23894, Cambridge, UK), CD105 (Abcam ab44967, Cambridge, UK) and MelanA (Abcam ab51061, Cambridge, UK) at 4°C overnight. Sections were washed and incubated with secondary antibody (Alexa-594 conjugated goat anti-mouse, 1∶250, Invitrogen A11032 for CD90 and CD105 and Alexa-594 conjugated goat anti-rabbit, 1∶250, Invitrogen A11037, Life technologies, Paisley, UK) for 1 hour at room temperature and mounted in Vectashield with DAPI (Vector laboratories Ltd. Peterborough, UK). Images were captured using a Zeiss 710 confocal microscope (Carl Zeiss, Hertfordshire, UK).
7
Immunofluorescence Assay for T. gondii
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T. gondii strains were used to infect coverslips with a confluent monolayer of HFFs under the indicated time constraints for the IFA analyses. The coverslips were fixed in 3.7% formaldehyde–phosphate-buffered saline (PBS) for 15 min and then blocked and permeabilized in 3% bovine serum albumin (BSA)–0.2% Triton X-100–PBS for 30 min. The samples were then incubated with primary antibody diluted in 3% BSA–0.2% Triton X-100–PBS for 1 h at room temperature. The coverslips were then washed in PBS (5 times for 5 min each) and treated with secondary antibodies Alexa 488-conjugated goat anti-mouse and/or Alexa 594-conjugated goat anti-rabbit (Molecular Probes) diluted 1:2,000 in 3% BSA–0.2% Triton X-100–PBS (27 (link), 52 (link)).
8
Immunofluorescent Labeling of Embryonic Tissues
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Immunohistofluorescence procedures were carried out in toto embryos as follows: (1) First incubation was conducted for 72 h at 4°C in the dilution of a mixture of primary antibodies (Table 3 ), combining mouse anti-Nkx2.2 with rabbit anti-GABA; and (2) Second incubations were conducted with Alexa 594-conjugated goat anti-rabbit (Molecular Probes, Eugene, OR; catalog reference A11037), and Alexa 488-conjugated goat anti-mouse (Molecular Probes; catalog reference A21042). Both secondary antibodies were diluted 1:500 in PB containing 0.5–1% Triton X-100, and incubation lasted 90 min at room temperature. Finally, the embryos were gelatin blocked (as detailed above) and cut on a freezing microtome at 14 μm in the transverse, horizontal, or sagittal plane. After being rinsed, the sections were mounted on glass slides and coverslipped with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA; cat. no. H1000).
9
Parkinson's Disease Animal Model Development
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Tween 80, 6-hydroxydopamine (6-OHDA), ascorbic acid (0.02%), paraformaldehyde, sucrose solutions, desipramine, and R-apomorphine hydrochloride were purchased from Sigma (MO, USA). Xylazine and Zoletil 50 were purchased from Bayer (Seoul, South Korea) and Vibac SA (Carros, France), respectively. Mouse anti-tyrosine hydroxylase (MAB5280), mouse anti-nestin (MAB353), and rabbit anti-GFAP-δ (AB9598) primary antibodies were purchased from EMD Millipore (MA, USA). Rabbit anti-laminin (L9393) and rabbit anti-TNF-α (ab66579) primary antibodies were obtained from Sigma (MO, USA) and Abcam (MA, USA), respectively. All secondary antibodies (Alexa 488-conjugated goat anti-rabbit, Alexa 594-conjugated goat anti-rabbit, and Alexa 594-conjugated goat anti-mouse IgG) were purchased from Molecular Probes (Invitrogen, CA, USA). Surgical sutures were obtained from Ethicon (4–0 Nylon; NJ, USA). Phosphate buffered saline (PBS) at pH 7.4 and 0.9% NaCl solution were obtained from Seoul National University Hospital Biomedical Research Institute (Seoul, South Korea) and Choongwae (Seoul, South Korea), respectively. Rodent chow was provided by Feedlab (Guri, South Korea). CoQ10 was a kind gift from Somang Cosmetics (Incheon, South Korea).
10
Morphine-Induced mTOR Phosphorylation in OPRM1 Variants
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