To immunostain cells or tissues (frozen sections), the samples were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature. After the cells were washed three times in PBS/0.1% BSA for 5 min, they were permeabilized using 0.2% Triton (Sigma, St Louis, MO, USA; T9284) in PBS for 20 min and then washed in PBS/0.1% BSA. Primary antibodies against CD73, SOX-9, RUNX-2, Coll-2, Coll-1, Ob-Rb, p53, p21 (all Abcam, Cambridge, UK) and CD146 (sc-18942; Santa Cruz, CA, USA) were diluted in PBS/0.1% BSA to 1/150 and incubated overnight at 4 °C. After the samples were washed, the cells or frozen sections were incubated with a FITC-conjugated goat anti-rabbit secondary antibody (1:500; Abcam) and DAPI (Sigma) for 1 h at room temperature. Fluorescent images were obtained using a Nikon A1-R (Melville, NY, USA) inverted confocal microscope.
Fitc conjugated goat anti rabbit secondary antibody
Manufactured by Abcam
Sourced in United Kingdom, Puerto Rico, China, United States
FITC-conjugated goat anti-rabbit secondary antibody is a laboratory reagent used to detect and visualize target proteins bound by primary rabbit antibodies. It contains fluorescein isothiocyanate (FITC) conjugated to a goat-derived antibody that binds to rabbit immunoglobulins.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (6 mentions)
Triton, by Merck Group (2 mentions)
T9284, by Merck Group (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Cd146, by Santa Cruz Biotechnology (1 mentions)
Coll 2, by Abcam (1 mentions)
Coll 1, by Abcam (1 mentions)
Ob rb, by Abcam (1 mentions)
Goat anti mouse igg secondary antibody, by Abcam (1 mentions)
Lsm 510 confocal laser scanning microscope, by Zeiss (1 mentions)
A1r inverted confocal microscope, by Nikon (1 mentions)
Ri1 color cooled camera system, by Nikon (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Anti e cadherin, by Abcam (1 mentions)
Anti vimentin, by Abcam (1 mentions)
Anti n cadherin antibody, by Abcam (1 mentions)
Anti foxo3a antibody, by Cell Signaling Technology (1 mentions)
Cy3 conjugated goat anti rabbit secondary antibody, by Bioss Antibodies (1 mentions)
Ix71 microscope, by Olympus (1 mentions)
Cytopainter f actin staining kit, by Abcam (1 mentions)
10 protocols using fitc conjugated goat anti rabbit secondary antibody
1
Immunostaining of Cellular Markers in Frozen Tissue Sections
2
Immunostaining of Ob-Rb Receptor in Cells
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To immunostain the cells or tissues (frozen sections), the samples were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 minutes at room temperature. After the cells were washed three times in PBS/0.1% bovine serum albumin (BSA) for five minutes, they were permeabilized using 0.2% Triton (T9284; Sigma-Aldrich) in PBS for 20 minutes and then washed in PBS/0.1% BSA. Primary antibodies against Ob-Rb (Abcam) were diluted in PBS/0.1% BSA and incubated overnight at 4°C. After the samples were washed, the cells or frozen sections were incubated with a fluorescein isothiocyanate (FITC)-conjugated goat antirabbit secondary antibody (1:500; Abcam), a goat antimouse IgG secondary antibody (1:500; Abcam), and 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for one hour at room temperature. Fluorescence images were obtained using a NikonEclipse TE2000-U inverted fluorescent microscope (Nikon, Tokyo, Japan).
3
Immunohistochemistry of S. argus Kidney
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Kidney tissue samples collected from S. argus were fixed for 16 h in 4% paraformaldehyde at 4 °C, dehydrated using a graded series of sucrose solutions, embedded in Tissue-Tek O.C.T (Sakura, Japan), and cut into 20-μm sections at −20 °C. Sections were mounted on poly-l -lysine-coated slides (ShiTai, China), incubated in 5% skim milk for 1 h at 37 °C, incubated with primary antibody (1:400 dilution) in PBS containing 0.5% Triton X-100 (PBST) overnight at 4 °C, then incubated with FITC-conjugated goat anti-rabbit secondary antibody (Abcam, UK) (1:1000 dilution) in PBST for 3–4 h at the room temperature. Nuclei were stained with DAPI (300 nM, Sigma-Aldrich, USA) in PBS for 5 min.
Cultured primary cells were fixed for 15 min in 4% paraformaldehyde, incubated in 5% skim milk for 0.5 h at 37 °C, incubated with primary antibody (1:400 dilution) in PBS containing 0.5% Triton X-100 (PBST) for 3 h at 4 °C, then with FITC-conjugated goat anti-rabbit secondary antibody (1:1000 dilution) in PBST for 1.5 h at room temperature. Nuclei were stained with DAPI (as above) for 3 min.
Samples were visualized with a Zeiss LSM510 confocal laser scanning microscope (Zeiss, Germany). Negative controls were incubated with normal rabbit serum.
Cultured primary cells were fixed for 15 min in 4% paraformaldehyde, incubated in 5% skim milk for 0.5 h at 37 °C, incubated with primary antibody (1:400 dilution) in PBS containing 0.5% Triton X-100 (PBST) for 3 h at 4 °C, then with FITC-conjugated goat anti-rabbit secondary antibody (1:1000 dilution) in PBST for 1.5 h at room temperature. Nuclei were stained with DAPI (as above) for 3 min.
Samples were visualized with a Zeiss LSM510 confocal laser scanning microscope (Zeiss, Germany). Negative controls were incubated with normal rabbit serum.
4
Immunostaining Cells for Col2 and Col1
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To immunostain cells, the samples were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature. After the cells were washed three times in PBS/0.1% BSA for 5 min, they were permeabilized using 0.2% Triton (Sigma; T9284) in PBS for 20 min, and then washed in PBS/0.1% BSA. Primary antibodies against Col2 and Col1 (Abcam, UK) were diluted 1/150 in PBS/0.1% BSA and incubated overnight at 4°C. After the samples were washed, the cells were incubated with a FITC-conjugated goat anti-rabbit secondary antibody (1:500; Abcam) and DAPI (Sigma) for 1 h at room temperature. Fluorescent images were obtained using a Nikon A1-R inverted confocal microscope.
5
Quantifying DNA Damage Response via γH2AX Foci
6
Immunofluorescence Staining of Cell Markers
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The indicated cells were cultured onto coverslips in 6-well plates until 70% confluence. Cells were then fixed with 4.0% paraformaldehyde in phosphate-buffered saline for 15 min, and blocked with 5% goat serum for 30 min. Next, the coverslips were incubated at 4°C with the indicated primary antibodies overnight. Anti-FOXO3a antibody was purchased from Cell Signaling Technology, Inc. Anti-E-cadherin and anti-Vimentin antibodies were purchased from Epitomics, Inc. Anti-N-cadherin antibody was purchased from Abcam, Inc. Subsequently, the coverslips were incubated with FITC-conjugated goat anti-rabbit secondary antibody (Abcam) or Cy3-conjugated goat anti-rabbit secondary antibody (Bioss, Beijing, P.R. China), dyed with Hoechst33342, and fixed in glycerol. The images were obtained with an Olympus IX71 microscope (Olympus, Tokyo, Japan), and color mergence was performed using ImageJ image software (ImageJ version 1.44p, NIH, MD).
7
Immunostaining of CRABP2 in Cells
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To immunostain cells, the samples were fixed in 4% paraformaldehyde in PBS for 40 min at room temperature. After the cells were washed 3 times in PBS/0.1% BSA for 5 min, they were permeabilized using 0.2% Triton (Sigma, St Louis, MO, USA; T9284) in PBS for 20 min and then washed in PBS/0.1% BSA. Primary antibodies against CRABP2 (Abcam, Cambridge, UK) were diluted in PBS/0.1% BSA at 4°C. After washing, the cells were incubated with a FITC-conjugated goat anti-rabbit secondary antibody (1: 500; Abcam) and DAPI (Sigma) for 1 h at 30°C. Fluorescent images were obtained using a Nikon microscope.
8
CXCR4 Expression in Neural Progenitor Cells
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NPSCs were seeded on slides and treated with or without 100 ng/mL SDF-1α for 24 h. For inhibition, the cells were pre-treated with 10 µg/mL AMD3100 for 2 h before treatment with 100 ng/mL SDF-1α. Then, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-X-100 in PBS, and blocked with 10% goat serum. Subsequently, the cells were incubated with primary antibody rabbit-anti-rat CXCR4 (1:50 dilution; Abcam) at 4 °C overnight. After PBS washing, cells were incubated with FITC-conjugated goat-anti-rabbit secondary antibody (Abcam) and red fluorescence-conjugate phalloidin working solution using a CytoPainter F-actin staining kit (Abcam) at room temperature for 60 min before counterstaining with DAPI (Beyotime, Shanghai, China). Cells that were incubated only with secondary antibody served as the negative control. Images were obtained using a confocal laser scanning microscope (LSM780, Zeiss, Germany).
9
Immunofluorescence analysis of IRF1 expression
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The treated cells were placed in 24-well plates with circular slides, up to 50-60% confluence fixed with 4% paraformaldehyde at room temperature for 10 min after X-ray radiation for 0, 3 and 6 h, and then treated with 1% Triton X-100 for 10 min at room temperature. After blocking with 1% BSA (Beyotime Institute of Biotechnology) for 1 h at room temperature, the samples were incubated with the IRF1 antibody (dilution, 1:1,000; cat. no. ab26109; Abcam) overnight at 4°C, and then incubated with FITC-conjugated goat anti-rabbit secondary antibody (dilution, 1:1,000; cat. no. A0562; Beyotime Institute of Biotechnology) for 2 h at room temperature. The nuclei were stained with DAPI (Sigma-Aldrich; Merck KGaA). The cells were observed under a confocal microscope (Olympus Corporation).
10
Immunofluorescence Staining of Fibroblasts
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The morphology and purity of prepared SFs were identi ed by detecting the expression of Vimention protein (the speci c marker of broblast cells) using immuno uorescence staining. SFs were washed with PBS three times and xed with 200 µL of methanol per well. Then the cells were washed with PBS, and incubated for 1 h in blocking solution containing 2% BSA (Beijing Zhongshan Jinqiao Biological Technology Co, Ltd, Beijing, China). The cells were incubated with vimentin antibodies (1: 500, Abcam, USA), followed by incubation with FITC-conjugated goat anti-rabbit secondary antibody (1:1000, Abcam, USA). Finally, the cells were counterstained with DAPI and visualized by BX51 positive immuno uorescence microscopy (OLYMPUS, Japan).
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