The sections were deparaffinized and 3% H2O2 was used to block the endogenous peroxidase for 30 min to inactivate the endogenous peroxidase. The sections were incubated in Tris-buffered saline (TBS) with 5% albumin bovine V (BSA) (Solarbio, A8020) for 1 h, and then incubated with a monoclonal anti-mouse α-SMA antibody (1:200, Abcam, Cambridge, MA, USA), anti-rabbit p53 antibody (1:200, Proteintech, USA), and anti-rabbit SFTPC antibody (1:100, Abcam, USA) in 5% BSA overnight at 4 °C. After washing with TBS, the sections were incubated with a FITC-conjugated goat anti-rabbit antibody (1:2000, Abcam, USA). The nuclei were counterstained with DAPI (Invitrogen, USA). The sections were then washed three times with PBS, coverslips mounted in 90% glycerol in PBS. The fluorescence detected by a fluorescence microscope (Nikon, Japan).
Fitc conjugated goat anti rabbit antibody
Manufactured by Abcam
Sourced in United States, United Kingdom, China
FITC-conjugated goat anti-rabbit antibodies are secondary antibodies that bind to primary rabbit antibodies. The FITC (fluorescein isothiocyanate) label allows for fluorescent detection. These antibodies can be used in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
2 7 dichlorodihydrofluorescein diacetate dcf da, by Merck Group (4 mentions)
Cytofix cytoperm buffer, by BD (3 mentions)
Apc cy7 conjugated streptavidin, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Rabbit anti γh2ax, by Cell Signaling Technology (2 mentions)
Cytoperm permeabilization buffer plus, by BD (2 mentions)
Anti mouse ly 6a ea sca 1 pe cy7, by Thermo Fisher Scientific (2 mentions)
Rabbit anti nox4, by Proteintech (2 mentions)
Cd45r b220, by Thermo Fisher Scientific (2 mentions)
Cd11b, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Sartorius (2 mentions)
Ter119, by Thermo Fisher Scientific (2 mentions)
Biotin conjugated cd5, by Thermo Fisher Scientific (2 mentions)
Rabbit anti nox4 antibody, by Proteintech (2 mentions)
Rabbit anti γh2ax antibody, by Cell Signaling Technology (2 mentions)
Ly6g gr 1, by Thermo Fisher Scientific (2 mentions)
Cd117 c kit apc, by Thermo Fisher Scientific (2 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Cell staining buffer, by BioLegend (1 mentions)
12 protocols using fitc conjugated goat anti rabbit antibody
1
Immunofluorescence Analysis of α-SMA, p53, and SFTPC
2
Histone H4 Expression in Blood Cells
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Citrated blood (20 μl) was diluted with 40 μl Cell Staining Buffer (BioLegend, San Diego, CA, USA). CTHs (final concentration 200 μg/ml) were added and samples were incubated for 30 min, 37°C, 5% CO2. Cells were washed in 900 μl Cell Staining Buffer, centrifuged (500 x g for 15 min, 4°C) and the pellet was resuspended in 200 μl buffer. Histone H4 bound to cells was stained with 2 μg/ml of a primary rabbit anti-human histone H4 (Abcam, Cambridge, UK) for 30 min on ice. Erythrocytes were lysed first with 200 μl Lysis buffer A (10 min, room temperature, dark) followed by 1 ml Lysis buffer B (10 min, room temperature, dark). Supernatant containing lysed erythrocytes were removed and remaining white blood cells were resuspended in 1 ml cold Cell Staining Buffer. A secondary FITC-conjugated goat anti-rabbit antibody (Abcam, Cambridge, UK) was added (2 μg/ml) and samples were incubated for 30 min on ice in dark. Two more wash steps were performed and each sample was analyzed in flow cytometry.
3
Immunofluorescent Staining of IPF Lung Tissue
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Paraffin blocks of IPF and control lung tissues were cut into 4-μm-thick slices, deparaffinized, rehydrated, and stained using hematoxylin and eosin. The sections were incubated in Fc receptor blocker (InnovexBiosciences, Richmond, CA, USA) for 30 min, incubated in TBS with 5% BSA for 1 h to block non-specific binding, and then incubated with monoclonal anti–human CCL8 antibody (1:100, Origene, Rockville, MD, USA) or polyclonal anti-human α-SMA antibody (1:200, Abcam, Cambridge, MA, USA) in 5% BSA overnight at 4 °C. After washing with 1 × TBS, the sections were incubated with secondary antibodies: FITC-conjugated goat anti-rabbit antibody (1:2000, Abcam) and PE-conjugated donkey anti-mouse antibody (1:2000, Abcam). Nuclei were counterstained with DAPI (Invitrogen, Carlsbad, CA, USA). Confocal laser scanning was performed using a microscope (LSM 510 META at 100× magnification) coupled to a Photometrics Coolsnap HQ camera (Photometrics, Tucson, AZ, USA), and images were generated using the Zeiss LSM image browser.
4
Targeting Hepatic Stellate Cells with cRGD-PLGA-Fe3O4-PFOB NPs
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HSCs were isolated from rats by collagenase digestion followed by OptiPrep density gradient centrifugation (Sun, et al., 2006 (link); Jeong, et al., 2006 (link); Horiguchi, et al., 2008 (link)). After 48 h, primary quiescent HSCs (qHSCs) showed features of myofibroblasts and were therefore identified as aHSCs. Primary rat HSCs cultured for 24 and 48 h were applied for further experiments. After incubation for 30 min with cRGD- PLGA- Fe3O4- PFOB NPs (25 mg/ml), cells were washed with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde, and incubated overnight with primary rabbit glial fibrillary acidic protein (GFAP, diluted 1:200, Abcam, United Kingdom) and primary rabbit α-smooth muscle actin (α-SMA, diluted 1:200, Abcam, United Kingdom) at 4°C. The secondary antibody selected for visualisation was FITC-conjugated goat anti-rabbit antibody (1:200; Abcam, United Kingdom). 4′6-diamindino-2-phenylindole (DAPI) was used to dye the cell nuclei. Finally, the ability of cRGD- PLGA- Fe3O4- PFOB NPs to target HSCs was observed via CLSM.
5
Immunostaining of Neural Stem Cell Markers
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Cells were xed 4% paraformaldehyde in 0.2M phosphate-buffered saline (PBS) for 20 min, were permeabilized in 0.3% Triton X-100 in PBS for 5 min and blocked with 10% normal goat serum for 10 min at room temperature, respectively. Then, cells were incubated with the appropriate antibody overnight at 4°C. The primary antibodies were rabbit anti-nestin (1:250; Abcam), rabbit anti-CD133 (1:500; Abcam), rabbit anti-GFAP (1:300, Abcam), rabbit anti-MAP2 (1:300, Abcam), rabbit anti-OLIG2 (1:300, Abcam). Following washing with PBS, the cells were incubated with secondary FITC-conjugated goat anti rabbit antibody (1:1000; Abcam) and FITC-conjugated goat anti mouse antibody (1:1000; Abcam) for 2 h at room temperature. Nuclei were stained with DAPI (blue). Finally, immunopositive cells were observed using inverted uorescence microscopy.
6
Cellular Oxidative Stress Analysis
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XMU‐MP‐1 was provided by Selleck chemicals; 2′, 7′‐Di‐chlorodi‐hydrofluorescein diacetate (DCFDA) and dichloro‐fluorescein (DCF) were obtained from Sigma. Anti‐mouse Ly‐6G/Ly‐6C (Gr‐1) was abtained from BD bioscience. Anti‐mouse CD117(c‐kit)‐APC, anti‐mouse Ly‐6A/EA(Sca‐1)‐PE/Cy7, biotin‐conjugated anti‐mouse CD45R/B220, anti‐mouse CD11b, anti‐mouse Ter‐119 and APC‐Cy7‐conjugated streptavidin were obtained from eBioscience. Rabbit anti‐γ‐H2AX was obtained from Cell Signaling Technology. Rabbit anti‐NOX4 was obtained from Proteintech. FITC‐conjugated goat anti‐rabbit antibodies were obtained from Abcam Biotechnology. Cytofix/Cytoperm buffer, Perm/Wash buffer and Cytoperm Permeabilization Buffer Plus were obtained from BD Pharmingen.
7
Assessing Immune Cell Apoptosis and Oxidative Stress
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The anti-mouse Ly-6A/EA (Sca-1)-PE/Cy7, CD117 (c-kit), APC, biotin-conjugated CD5, CD4, CD8, CD45R/B220, Ly6G/Gr-1, CD11b, Ter-119, and APC/CY7-conjugated streptavidin antibodies, and an Annexin V-FITC apoptosis kit were purchased from eBioscience (San Diego, CA, USA). The 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA), α-lipoic acid, and 5-methoxytryptamine were purchased from Sigma-Aldrich (St. Louis, MO, USA). The RPMI 1640 medium was purchased from Gibco (Grand Island, NY, USA). The BD Cytofix/Cytoperm buffer was purchased from BD Biosciences (San Diego, CA, USA). Methylcellulose M3534 was purchased from Stem Cell (Vancouer, BC, Canada). Fetal calf serum was purchased from Biological Industries (Kibbutz, Israel). The rabbit anti-γH2AX antibody was obtained from Cell Signaling Technology (Danvers, MA, USA), the rabbit anti-NOX4 antibody from Proteintech (Wuhan, China) and the FITC-conjugated goat anti-rabbit antibodies from Abcam (Cambridge, MA, USA).
The 5-methoxytryptamine-α-lipoic acid (MLA) was synthetized using α-lipoic acid and 5-methoxytryptamine at the drug department of the Institute of Radiation Medicine, CAMS. Melatonin (MLT) was purchased from Tokyo Chemical Industry Co., Ltd. (Shanghai, China). MLA and MLT were dissolved in a 5% carboxymethyl cellulose (CMC) solution.
The 5-methoxytryptamine-α-lipoic acid (MLA) was synthetized using α-lipoic acid and 5-methoxytryptamine at the drug department of the Institute of Radiation Medicine, CAMS. Melatonin (MLT) was purchased from Tokyo Chemical Industry Co., Ltd. (Shanghai, China). MLA and MLT were dissolved in a 5% carboxymethyl cellulose (CMC) solution.
8
Multiparametric analysis of murine cells
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A formulation of XH-201 and XH-202 was used in these studies. The following antibodies were purchased from eBioscience (San Diego, CA, USA): anti-mouse Ly-6A/EA (Sca-1)-PE, rat IgG2a- PE (Isotype Ctrl), CD117 (c-kit)-APC, rat IgG2b-APC (Isotype Ctrl), biotin-conjugated CD5, CD4, CD8, CD45R/B220, Ly6G/Gr-1, CD11b, Ter-119 and PerCP-conjugated streptavidin antibodies [20 (link)]. RPMI 1640 medium was purchased from Gibco (Grand Island, NY, USA). Melatonin was purchased from Tixiai Chemical Industry Co., Ltd (Shanghai, China). BD Cytofifix/Cytoperm buffer was purchased from BD Biosciences (San Diego, CA, USA). MitoSOX red mitochondrial superoxide indicator was obtained from Life Technologies (Grand Island, NY, USA). Rabbit anti-γH2AX antibody was obtained from Cell Signaling Technology (Danvers, MA, USA) and FITC-conjugated goat anti-rabbit antibodies from Abcam (Cambridge, MA, USA).
9
Multiparameter Immune Cell Analysis
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The anti-mouse Ly-6A/EA (Sca-1)-PE/Cy7, CD117 (c-kit), APC, biotin-conjugated CD5, CD4, CD8, CD45R/B220, Ly6G/Gr-1, CD11b, Ter-119, and APC/CY7-conjugated streptavidin antibodies were purchased from eBioscience (San Diego, CA, USA). The 21, 71-dichlorodihydrofluorescein diacetate (DCFDA) was purchased from Sigma-Aldrich (St Louis, MO, USA). The RPMI 1640 medium was purchased from Gibco (Grand Island, NY, USA). The BD Cytofix/Cytoperm buffer was purchased from BD Biosciences (San Diego, CA, USA). Methylcellulose M3434 was purchased from Stem Cell (Vancouer, BC, Canada). Fetal calf serum was purchased from Biological Industries (Kibbutz, Israel). The rabbit anti-H2AX antibody was obtained from Cell Signaling Technology (Danvers, MA, USA), the rabbit anti-NOX4 antibody from Proteintech (Wuhan, China) and the FITC-conjugated goat anti-rabbit antibodies from Abcam (Cambridge, MA, USA). Hei brick tea was purchased from Baishaxi Tea Industry (Hunan, China). Malondialdehyde (MDA), SOD, CATand GSHPx reagent kits were purchased from Nanjing Jiancheng Bioengineering (Jiangsu, China).
10
Murine Immune Cell Phenotyping and Oxidative Stress Analysis
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Eight- to 10-week-old male C57BL/6J mice were purchased from Vital River (Beijing, China), mice were housed in the Specific Pathogen Free level animal facility at the Institute of Radiation Medicine (IRM), the Chinese Academy of Medical Sciences (CAMS). All experimental procedures were approved by the Institutional Animal Care and Use Committee of the CAMS (Permit Number 1526, 7 April 2015), and written informed consent was obtained from all participants.
SB203580 was purchased from LC Laboratories (Boston, MA, USA). Biotin-conjugated anti-Mouse-CD4 (clone 34 GK1.5), anti-Mouse-CD8 (clone 53-6.7), anti-mouse-CD11b (clone M1/70), anti-mouse-CD45R/B220 (clone RA3-6B2), anti-mouse-Ly6G/Gr-1 (clone RB6-8C5), anti-mouse-Ter-119 (clone Ter-119), anti-mouse-CD117 (c-kit)-APC (clone 2B8), anti-mouse-Ly-6A/EA (Sca-1)-PE/Cy7 (clone D7), and APC-Cy7-conjugated streptavidin were purchased from eBioscience (San Diego, CA, USA). 2,7-dichlorodihydrofluorescein diacetate (DCFDA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-p-p38 (ab61241), rabbit anti-p16, and FITC-conjugated goat anti-rabbit antibodies were obtained from Abcam Biotechnology (Cambridge, MA, USA).
SB203580 was purchased from LC Laboratories (Boston, MA, USA). Biotin-conjugated anti-Mouse-CD4 (clone 34 GK1.5), anti-Mouse-CD8 (clone 53-6.7), anti-mouse-CD11b (clone M1/70), anti-mouse-CD45R/B220 (clone RA3-6B2), anti-mouse-Ly6G/Gr-1 (clone RB6-8C5), anti-mouse-Ter-119 (clone Ter-119), anti-mouse-CD117 (c-kit)-APC (clone 2B8), anti-mouse-Ly-6A/EA (Sca-1)-PE/Cy7 (clone D7), and APC-Cy7-conjugated streptavidin were purchased from eBioscience (San Diego, CA, USA). 2,7-dichlorodihydrofluorescein diacetate (DCFDA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-p-p38 (ab61241), rabbit anti-p16, and FITC-conjugated goat anti-rabbit antibodies were obtained from Abcam Biotechnology (Cambridge, MA, USA).
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