Liver tissues were cryo-sectioned and processed for the immunofluorescence (IF) assay as described previously (Alam et al., 2012 (link)). RBC-EM DiD or PBS-injected mouse liver sections were stained with anti-rabbit CD68 (Abcam), followed by staining with goat anti-rabbit FITC (Abcam). Tissue sections were mounted using the VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA, United States). IF-stained sections were imaged under a confocal microscope (LSM 5 Exciter, Zeiss, Oberkochen, Germany). Total of six fields were counted by observers. Number of CD68 positive (CD68 +; green) cells were counted from RBC-EM DiD or PBS-injected mouse liver sections. Then the number of DiD positive (DiD +; red) with CD68 + or CD68 - cells were counted.
Goat anti rabbit fitc
Manufactured by Abcam
Sourced in United Kingdom
Goat anti-rabbit FITC is a secondary antibody conjugated with the fluorescent dye FITC (Fluorescein isothiocyanate). It is designed to detect and bind to rabbit primary antibodies in various immunoassays and imaging techniques.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 5 exciter, by Zeiss (2 mentions)
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Effluc, by Promega (1 mentions)
Cd16 32, by BD (1 mentions)
Cd24 pe clone m1 69, by BD (1 mentions)
Facsaria 3, by BD (1 mentions)
Fortessa, by BD (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Kim 1, by Abcam (1 mentions)
Sirt1, by Abcam (1 mentions)
A1r laser scanning confocal microscope, by Nikon (1 mentions)
6 protocols using goat anti rabbit fitc
1
Immunofluorescence Imaging of Liver Macrophages
2
Immunofluorescence Analysis of Mouse Tumor Tissues
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Tumor tissues were cryo-sectioned and processed for immunofluorescence assay as described previously58 (link). CAL-62/Rluc- or PBS-injected mouse tumor sections were stained with Rluc (Abcam; Dilution: 1:400), followed by goat anti-rabbit FITC (Abcam; Dilution: 1:500); Effluc (Promega; Dilution: 1:300), and donkey anti-goat Alexa Fluor® 647 (Abcam; Dilution: 1:500). Tissue sections were mounted using VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA, USA). IF-stained sections were imaged under a confocal microscope (LSM 5 Exciter, Zeiss, Oberkochen, Germany).
3
Flow Cytometry Analysis of Cell Surface Mmp14
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Cells were resuspended in FACS buffer (PBS, 2% FCS and 5 mM EDTA) and stained at a concentration of 1 × 104 cells/μl. After blocking of unspecific antibody binding with CD16/32 (BD Pharmingen, Franklin Lakes, NJ, USA), specific antibodies were added at appropriate dilutions (CD24PE, clone M1/69, BD Pharmingen; CD90PE-Cy5, clone HIS51, eBioscience, San Diego, CA; CD45PE-Cy7, clone 30-F11, eBioscience) for 30 min on ice in the dark, followed by addition of 4′,6-Diamidin-2-phenylindol (DAPI). Stained cells were then washed and resuspended in 200-1000 μl FACS buffer. For Mmp14 staining, cells were first incubated with the primary antibody, anti-Mmp14, clone EP1264Y, and subsequently with the secondary antibody goat-anti rabbit FITC, both Abcam, Cambridge, UK, at appropriate dilutions.
Sorting and analysis of cells was performed using a FACSAriaIII or Fortessa flow-cytometer, with Diva (BD Bioscience, Franklin Lakes, NJ, USA) and FlowJo (FlowJo, LLC, Ashland, OR, USA) software. Forward and side scatter profiles, depletion of DAPI positive, as well as CD45 positive cells were used as selection criteria. Cells that were either positive for both CD24 and CD90 or negative for either or both markers were analyzed for Mmp14 cell surface expression, and collected.
Sorting and analysis of cells was performed using a FACSAriaIII or Fortessa flow-cytometer, with Diva (BD Bioscience, Franklin Lakes, NJ, USA) and FlowJo (FlowJo, LLC, Ashland, OR, USA) software. Forward and side scatter profiles, depletion of DAPI positive, as well as CD45 positive cells were used as selection criteria. Cells that were either positive for both CD24 and CD90 or negative for either or both markers were analyzed for Mmp14 cell surface expression, and collected.
4
Immunocytochemistry Analysis of hUMSCs
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12 days after treatment with RA or co-culture, hUMSCs were rinsed with PBS and fixed in 4% PFA for 10 min at room temperature.
In order to accomplish permeabilization, cells were washed with PBS 3 times and incubated in 0.1% Triton X-100 [Sigma, Missouri, United States] for 10 min. For blocking non-specific binding sites, cells were incubated in blocking solution, including PBS with 5% goat serum for 45 min at room temperature. Afterward, cells were incubated with anti-NSE, anti-MAP2, and anti-ChAT antibodies [Abcam, Cambridge, United Kingdom] overnight at 4 °C, followed by incubation with Goat Anti-Rabbit FITC [Abcam, Cambridge, United Kingdom] in PBS with 1% FBS for 1 hour at room temperature. For crosslinking antigen/antibody complexes, cells were washed with PBS and fixed with 4% PFA for 5 min. Finally, cell nuclei were counter-stained with Propidium iodide (PI) [Sigma, Missouri, United States] for three minutes and examined under a fluorescent microscope (Olympus, IX71).
In order to accomplish permeabilization, cells were washed with PBS 3 times and incubated in 0.1% Triton X-100 [Sigma, Missouri, United States] for 10 min. For blocking non-specific binding sites, cells were incubated in blocking solution, including PBS with 5% goat serum for 45 min at room temperature. Afterward, cells were incubated with anti-NSE, anti-MAP2, and anti-ChAT antibodies [Abcam, Cambridge, United Kingdom] overnight at 4 °C, followed by incubation with Goat Anti-Rabbit FITC [Abcam, Cambridge, United Kingdom] in PBS with 1% FBS for 1 hour at room temperature. For crosslinking antigen/antibody complexes, cells were washed with PBS and fixed with 4% PFA for 5 min. Finally, cell nuclei were counter-stained with Propidium iodide (PI) [Sigma, Missouri, United States] for three minutes and examined under a fluorescent microscope (Olympus, IX71).
5
Immunofluorescence Staining of HK-2 Cells
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We used 24-well plates to culture HK-2 cells and added cell slides to the culture dishes. After treating the cells, we took out the culture dish and discarded the medium. We then fixed the cells with 4% paraformaldehyde for 10 minutes and continued incubation with 0.2% Triton-PBS for 15 minutes. After washing the cells with PBS, we blocked the cells with 10% goat serum for 30 minutes. Then, we discarded the liquid in the culture dish and added the primary antibody dilution (KIM-1, 1: 500, rabbit, Abcam, Cambridge, MA, USA; NGAL, 1: 500, rabbit, Abcam, Cambridge, MA, USA; Sirt1, 1: 500, rabbit, Abcam, Cambridge, MA, USA). The cells were incubated overnight at 4°C. The next day, after washing the cells with PBS, we incubated the cells with fluorescent secondary antibody dilution (Goat anti-rabbit-FITC, 1: 500, Abcam, Cambridge, MA, USA) for 1 hour and washed the cells. Then, we fixed the cells to the slides using a 4’,6-diamidino-2-phenylindole (DAPI)-containing capsule and observed the staining results using a fluorescence microscope.
6
Immunofluorescence Assay for DNA Damage Response
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For the immunofluorescence assay, cells cultured on coverslips were fixed with 4% paraformaldehyde for 15 min at room temperature, and washed with PBS for three times. Then the fixed cells were permeabilized with 0.25% Triton X-100 for 10 min, followed by 10 min-PBS washing for three times. Then the cells were blocked with 1% goat serum for 1 hr at room temperature, followed by the incubation with the indicated antibodies including anti-γH2AX (Cell signaling technology, Cat. #9718S), anti-53BP1 (Cell signaling technology, Cat. #4937S), anti-RAD51 (Abcam, Cat. #ab88572) or anti-RPA2 overnight at 4 °C. After washing with PBS for three times, the cells were incubated with the secondary antibody (Abcam, goat-anti-rabbit-FITC, Cat. #ab6717) for 1 hr at room temperature. Cells were then washed with PBS for three times before covered with mounting medium with DAPI (Abcam, Cat. #ab104939). The images were taken on a Nikon A1R laser scanning confocal microscope and analyzed with ImageJ.
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