MSCs or ESCs were cultured on gelatin-coated (Sigma, Dorset, UK) coverslips with or without the addition of 100 ng/ml equine IFN-γ (R + D Systems, Abington, UK) for 72 hours, fixed in 3% paraformaldehyde (in PBS) for 20 minutes at room temperature, and permeabilized for 1 hour with 0.1% Triton-X-100 at room temperature. Primary antibody incubations with mouse anti-MHCI 1:200 and mouse anti-MHCII 1:200 (both VMRD, Pullman, WA, USA) were carried out overnight at 4°C before detection with a secondary antibody goat anti-mouse FITC 1:200 (Abcam, Cambridgeshire, UK). Three replicates for each cell type were performed by using cells isolated from different animals.
Goat anti mouse fitc
Manufactured by Abcam
Sourced in United Kingdom, United States
Goat anti-mouse FITC is a secondary antibody conjugated with the fluorescent dye fluorescein isothiocyanate (FITC). It is used to detect and visualize mouse primary antibodies in various immunoassay techniques, such as immunohistochemistry, flow cytometry, and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Mouse monoclonal anti olig2, by Abcam (2 mentions)
Goat anti rabbit fitc, by Merck Group (2 mentions)
Gelatin coated, by Merck Group (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Act 1, by Nikon (1 mentions)
Nestin, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg fitc, by Abcam (1 mentions)
Glial fibrillary acidic protein (gfap), by Merck Group (1 mentions)
Fluorescent microscopy, by Olympus (1 mentions)
8 ohdg, by Abcam (1 mentions)
Mouse anti cd31, by Abcam (1 mentions)
Anti mouse f4 80, by Abcam (1 mentions)
Anti mouse cd206, by Abcam (1 mentions)
Sodium citrate buffer, by Thermo Fisher Scientific (1 mentions)
Edta buffer, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonalanti mbp, by Abcam (1 mentions)
Sucrose, by Merck Group (1 mentions)
Anti olig2 antibody, by Abcam (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti ubiquitin, by Santa Cruz Biotechnology (1 mentions)
13 protocols using goat anti mouse fitc
1
MHC Expression in Stem Cells
2
Quantifying Myelinating Oligodendrocytes in Rat Spinal Cord
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At the end of experiment, following deep anesthesia and fixative perfusion, rat spinal cord was dissected and processed for serial frozen sections (10 μM thick) using a cryostat microtome. In order to examine the presence of myelinating oligodendro-cyte, immunohistochemistry staining was carried out with specific antibody (16 (link)). Briefly, after sample permeabilization, incubation was performed in primary antibodies; mouse monoclonal anti-Major basic protein (MBP) (1:1000) (Abcam, Cambridge City, MA, USA) and mouse monoclonal anti-Olig2 (1:1000, Abcam), and subsequently in secondary antibody; Goat anti-mouse FITC - (1:2000, Abcam). Finally, nuclear counterstaining was performed using 4’, 6-Diamidino-2-Phenylindole, Dilactate (DAPI). The cells were examined using a fluorescence microscope (Olympus BX51, Tokyo City, Japan) for at least five sections of each sample. The ratio number of labeled positive cells to the number of nuclei was calculated for each antigen by Image J, a public domain, Java-based image processing program developed at the National Institutes of Health (NIH).
3
Immunofluorescence Assay for Picornavirus Detection
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IFA was conducted essentially according to the protocol outlined on the Abcam website (www.abcam.com ). SK-RST cells with over 90% confluency were infected with the reference strains of PTVs (Table 1 ) at an MOI of 0.1 for 20 h. After fixing with methanol for 15 min at −20 °C, the cells were washed and then incubated for 1 h at RT with the antisera or mouse anti-PTV mAb 040/4B1 (1:1000) (a gift from Dr. M. Dauber, Friedrich-Loeffler Institute, Federal Research Institute for Animal Health, Insel Riems, Germany) in PBS plus 0.1% BSA. Finally, the cells were incubated with goat anti-mouse-FITC (1:5000; Abcam) in PBS for 30 min at RT. Fluorescence was examined by an inverted microscope (Olympus, Japan) and was documented using the Nikon ACT-1 software.
4
Immunophenotyping of Adult Neural Stem Cells
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Immunofluorescence assay was done to identify the expression of markers in the isolated cells. After discarding the cell growth medium, fixation was carried out with 4% paraformaldehyde in PBS for 20 min at room temperature. Permeabilization of cells was conducted using 0.2% Triton X-100, and 5% normal goat serum (NGS; Abcam). Then, the cells were exposed to the primary antibody against the glial fibrillary acidic protein (GFAP) (1:200 diluted in PBS; Millipore), nestin (1:50 diluted in PBS; Santa Cruz), and Sox2 (1:100 diluted in PBS; Santa Cruz) as markers of adult neural stem/progenitor cells. Subsequently, the cells were incubated with secondary antibodies, including goat anti-rabbit IgG (FITC, 1:1000 diluted in PBS; Abcam), goat anti-mouse (FITC, 1:1000 diluted in PBS; Abcam), and rabbit anti-goat (FITC, 1:1000 diluted in PBS; Sigma) for 2 h at room temperature in the dark. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Cells were photographed using fluorescent microscopy (Olympus, Japan). Samples were incubated with secondary antibodies alone as negative control.
5
Bystander Killing Effect of MW2821 and EV
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To measure a bystander killing effect of 9MW2821 and EV, PC-3 and PC-3/nectin-4 cell lines were used as nectin-4–negative and positive cells, respectively. For the coculture study, PC-3 cells were seeded in a 6-well plate at fixed 3 × 104 cells/well, and PC-3/nectin-4 cells were seeded at 0, 0.3 × 104, 3 × 104, and 6 × 104 cells/well. After overnight incubation, the supernatant was removed, and each drug, MW282 mAb, 9MW2821, and EV, was added to 1 μg/mL (no added drug was the control). After 5 days of coculture, the supernatant was removed, and viable cells were detached from the plate. The number of cells was measured with a cell counter. To determine the ratio of PC-3 and PC-3/nectin-4 cells, the cells were stained with a mouse monoclonal anti-nectin–4 antibody (Clone# M008, prepared by Mabwell) on ice for 60 minutes. After washing, the cells were stained with goat anti-mouse FITC (Abcam, catalog No. ab150113) on ice for 60 minutes. After washing, the fluorescent signals of 1 × 104 stained cells were measured using a flow cytometer, and the ratio of nectin-4–positive to nectin-4–negative cells was calculated. The number of PC-3 or PC-3/nectin-4 cells in each well was calculated, and the bystander killing effect was evaluated on the basis of the number of PC-3 cells.
6
Wound Healing Analysis in Diabetic Mice
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Mice were sacrificed after 15 days. The wound tissues were fixed in 10% formalin and embedded in the paraffin routinely. The sections of diabetic wounds were stained with hematoxylin and eosin (H & E), and Masson’s trichrome. Images were scanned by ServiceBio Company. Picrosirius red staining pictures were photographed by polarized light microscope in Servicebio Company. The collagen content was quantified using ImageJ v1.8.0 software.
For immunostaining, wound sections were dewaxed and rinsed with PBS. For antigen retrieval, tissue sections were treated with sodium citrate buffer at 95 °C for 10 min, followed by permeabilization with 0.1% Triton X-100 for 10 min. After blocking in PBST (PBS + 0.1% Tween 20) containing 2% BSA, tissue sections were incubated with 8-OHdG (Abcam; 1:200), anti-mouse CD31 (Abcam; 1:300), anti-CK10 (Abcam; 1:100), anti-mouse F4/80 (Abcam; 1:100), anti-mouse CD86 (Abcam; 1:100) or anti-mouse CD206 (Abcam; 1:100) antibodies for 8 h at 4 °C. Goat anti-mouse FITC (Abcam;1:200) and goat anti-rabbit TRITC secondary antibodies (Abcam; 1:400) were added for 2 h at room temperature. The fluorescence pictures were collected through a confocal fluorescence microscope (Leica SPII) with 20× magnification.
For immunostaining, wound sections were dewaxed and rinsed with PBS. For antigen retrieval, tissue sections were treated with sodium citrate buffer at 95 °C for 10 min, followed by permeabilization with 0.1% Triton X-100 for 10 min. After blocking in PBST (PBS + 0.1% Tween 20) containing 2% BSA, tissue sections were incubated with 8-OHdG (Abcam; 1:200), anti-mouse CD31 (Abcam; 1:300), anti-CK10 (Abcam; 1:100), anti-mouse F4/80 (Abcam; 1:100), anti-mouse CD86 (Abcam; 1:100) or anti-mouse CD206 (Abcam; 1:100) antibodies for 8 h at 4 °C. Goat anti-mouse FITC (Abcam;1:200) and goat anti-rabbit TRITC secondary antibodies (Abcam; 1:400) were added for 2 h at room temperature. The fluorescence pictures were collected through a confocal fluorescence microscope (Leica SPII) with 20× magnification.
7
Quantitative Analysis of Oligodendrocyte Precursor Cells
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After tissue processing by use of ethanol, xylene
and paraffin (Asia Pajohesh, Iran), serial sections (5-
μm thick) of the brain samples were prepared using a
microtome. After de-paraffinization and rehydration,
the slices were pre-treated using the method of heatinduced
epitope retrieval with sodium citrate buffer
(pH=6) and 1 mM EDTA buffer (pH=8) (Gibco,
USA) for 20 minutes, washed-out three times with
PBS (Gibco, USA). Then, the slices were incubated
with special antibodies against A2B5, a marker of
oligodendrocyte precursor cells (Abcam, USA) and
MOG, a myelin oligodendrocyte glycoprotein (Abcam,
USA), and then washed-out with PBS. Then, goat antimouse
-FITC (2 μg/ml, Abcam, USA) and rabbit antigoat-
FITC (Sigma-Aldrich, USA) were used as the
secondary antibody at room temperature for 1 hour.
Finally, nuclear counterstaining was conducted using
4, 6 Diamino-2-phenylindole, dilactate (DAPI) and
in order to quantitative analysis, the total numbers
of positive cells were counted in a minimum total of
200 cells per slide in six sections per sample using
.uorescence microscope (Olympus bx51, Japan).
Meanwhile, all IHC analyses were repeated at least
three times.
and paraffin (Asia Pajohesh, Iran), serial sections (5-
μm thick) of the brain samples were prepared using a
microtome. After de-paraffinization and rehydration,
the slices were pre-treated using the method of heatinduced
epitope retrieval with sodium citrate buffer
(pH=6) and 1 mM EDTA buffer (pH=8) (Gibco,
USA) for 20 minutes, washed-out three times with
PBS (Gibco, USA). Then, the slices were incubated
with special antibodies against A2B5, a marker of
oligodendrocyte precursor cells (Abcam, USA) and
MOG, a myelin oligodendrocyte glycoprotein (Abcam,
USA), and then washed-out with PBS. Then, goat antimouse
-FITC (2 μg/ml, Abcam, USA) and rabbit antigoat-
FITC (Sigma-Aldrich, USA) were used as the
secondary antibody at room temperature for 1 hour.
Finally, nuclear counterstaining was conducted using
4, 6 Diamino-2-phenylindole, dilactate (DAPI) and
in order to quantitative analysis, the total numbers
of positive cells were counted in a minimum total of
200 cells per slide in six sections per sample using
.uorescence microscope (Olympus bx51, Japan).
Meanwhile, all IHC analyses were repeated at least
three times.
8
Assessing Myelination in Spinal Cord Transplants
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At the endpoint of experiment, the rats were
anesthetized and fixing process was performed through
transcardially perfusion methods with ice-cold PBS and
4% paraformaldehyde (PFA) in PBS (pH=7.4). Rat spinal
cord was removed and postfixed in the same fixative at 4°C
overnight. Then, the sample was cryoprotected by 30%
sucrose (Sigma-Aldrich, USA) in PBS for 48-72 hours.
Subsequently, serial frozen sections (10 µ thick) of the
spinal cords were prepared using a microtome cryostat. In
order to evaluate the presence of myelin forming cells in
transplantation area, immunofluorescence technique was
done with primary antibodies include mouse monoclonal
anti-MBP (1:1000), mouse monoclonal anti-Olig2
(1:1000) and Goat anti-mouse FITC (1:2000, all purchased
from Abcam, UK) as secondary antibody. Finally,
after labeling the cell nucleus using 4', 6-Diamidino2-
Phenylindole, Dilactate (DAPI) cells were observed
using a fluorescence microscope, and immunopositive
cells was counted in a minimum total of 200 cells per
slide. Meanwhile, all immunofluorescence studies were
repeated at least twice.
anesthetized and fixing process was performed through
transcardially perfusion methods with ice-cold PBS and
4% paraformaldehyde (PFA) in PBS (pH=7.4). Rat spinal
cord was removed and postfixed in the same fixative at 4°C
overnight. Then, the sample was cryoprotected by 30%
sucrose (Sigma-Aldrich, USA) in PBS for 48-72 hours.
Subsequently, serial frozen sections (10 µ thick) of the
spinal cords were prepared using a microtome cryostat. In
order to evaluate the presence of myelin forming cells in
transplantation area, immunofluorescence technique was
done with primary antibodies include mouse monoclonal
anti-MBP (1:1000), mouse monoclonal anti-Olig2
(1:1000) and Goat anti-mouse FITC (1:2000, all purchased
from Abcam, UK) as secondary antibody. Finally,
after labeling the cell nucleus using 4', 6-Diamidino2-
Phenylindole, Dilactate (DAPI) cells were observed
using a fluorescence microscope, and immunopositive
cells was counted in a minimum total of 200 cells per
slide. Meanwhile, all immunofluorescence studies were
repeated at least twice.
9
Oligodendrocyte Differentiation Immunofluorescence
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In the following oligodendrocyte differentiation, differentiated cells were fixed with 4% Paraformaldehyde (PFA) for 15 min at room temperature, incubated in 1% BSA/10% normal goat serum/0.3 M glycine in 0.1% PBS-Tween for 1 hr to permeabilise the cells and block non-specific protein-protein interactions. In the following, incubation with primary antibodies diluted in PBS with 0.1% BSA, overnight at 4°C in humidified condition was done. The following antibodies were used: anti-A2B5 antibody, 1 μg/ml; Abcam; anti-olig2 antibody, 1:1000; Abcam. After cell washing, the slides were treated with goat anti-mouse FITC (1:500; Abcam, UK)-conjugated secondary antibodies diluted in PBS with 0.1% BSA at RT for 1 hr. After this time, cells were washed and fixed with 4% PFA for 5 min at room temperature. Finally, the nuclei were stained with DAPI for cell counting using fluorescence microscope (Olympus, BX51, Japan). For quantitative analysis, the number of A2B5, olig2 positive cells was counted on each acquired image in a minimum total of 200 cells per slide.
10
Immunofluorescence Staining of hASCs
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hASCs cultured alone and co-cultured with SAOS2 and MCF7 cells were fixed with 4% paraformaldehyde, permeabilized with TRITON X-100 and blocked with bovine serum albumin at 5% for 1 h at room temperature and then stained with primary antibodies at 4 °C overnight. The primary antibody used was mouse anti-human vimentin (Invitrogen, Life Technologies Italia). The secondary antibody, goat anti-mouse FITC (Abcam, Cambridge, UK) diluted 1:200 in PBS, was incubated for 60 min at 4 °C, and the Hoechst33342 (Invitrogen, Life Technologies Italia) was used to stain the nucleus and was incubated for 7 min at room temperature. Cells were observed under the fluorescence microscope (EVOS, Life Technologies, Milan, Italy). Isotypes and non-probed cells were used as controls.
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