PKR tissue localization was examined on 8 μm cryosections (on day 7 after laser photocoagulation). The cryosections were blocked with 1% bovine serum albumin (BSA) for 4 h at room temperature and then incubated with antibodies for PKR (1:50; Santa Cruz Biotechnology) and CD31 (1:50, Abcam, Cambridge, MA) at 4 °C overnight. For CD31 staining, antigen retrieval was performed by incubating the sections in a heated water bath at 37 °C for 10 min. Thereafter, the slides were stained with Alexa Fluor 488 conjugated goat anti-mouse immunoglobulin G, Alexa Fluor 546 conjugated goat anti-rabbit IgG (1:200; Invitrogen), and Hoechst (1:2,000; Sigma-Aldrich). The photomicrographs were obtained using a digital high-sensitivity camera (Hamamatsu, ORCA-ER C4742–95; Hamamatsu, Japan).
Alexa fluor 488 conjugated goat anti mouse immunoglobulin g
Manufactured by Thermo Fisher Scientific
Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin G is a secondary antibody that binds to mouse immunoglobulin G (IgG) and is labeled with the Alexa Fluor 488 fluorescent dye. This product is commonly used in immunofluorescence techniques to detect and visualize mouse IgG-containing proteins or cells.
Automatically generated - may contain errors
Lab products found in correlation
Cellquest software, by BD (2 mentions)
Cellstripper, by Corning (2 mentions)
Anti αvβ3 and anti αvβ5 integrin antibodies, by Merck Group (2 mentions)
Desmoglein 2 dsg2 clone ah12, by Santa Cruz Biotechnology (2 mentions)
Facscan, by BD (2 mentions)
Orca er c4742 95, by Hamamatsu Photonics (1 mentions)
Alexa fluor 546 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Merck Group (1 mentions)
Casp3, by Abcam (1 mentions)
Il 1β, by Abcam (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
6 protocols using alexa fluor 488 conjugated goat anti mouse immunoglobulin g
1
Immunofluorescent detection of PKR in retinal tissue
2
Quantification of Integrin Expression
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Cell lines were detached from a 75-cm2 tissue culture flask by treatment with cell stripper (Cellgro; Manassas, VA.). Cells were counted, blocked and aliquots of 1.5 × 106 cells were incubated with 1.5 μg of anti-αVβ3 and anti-αVβ5 integrin antibodies (Millipore; Temecula, CA), 1.5 μg of mouse monoclonal antibody Desmoglein 2 (DSG2) (clone AH12.2, Santa Cruz Biotechnology Inc, Santa Cruz, CA) or untreated (control). Following primary antibody incubation, the cells were washed and incubated with Alexa fluor 488-conjugated goat anti-mouse immunoglobulin G (Invitrogen) diluted in PBS with 0.1% BSA. Cells were then diluted in PBS and a cytometric analysis of 10,000 events per sample was conducted using FACScan with CellQuest software (Becton Dickinson, Mountain View, CA).
3
CAR Antibody Expression Analysis
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Cells were cultured in a 75-cm2 tissue culture flask, harvested with PBS [pH 7.4] containing 0.53 mM EDTA, and washed with wash buffer (PBS [pH 7.4] containing 0.1% bovine serum albumin [BSA]). We incubated 5.0×104 cells with anti-human CAR monoclonal antibody RmcB [47] (link) which was kindly provided from Dr. Douglas [48] (link) for 1 hour. Following incubation, the cells were washed three times with wash buffer and incubated with Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin G (Invitrogen). After two additional washing steps, the cells were analyzed using MACSQuant Analyzer (Milteryl Biotec Inc. Auburn, CA) or Becton Dickinson Fluorescence Activated Cell Sorter (FACS aria) (BD Biosciences, Franklin Lakes, NJ).
4
Flow Cytometry Analysis of Integrin and Desmoglein Expression
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Example 5
Flow Cytometry:
Cell lines were detached from a 75-cm2 tissue culture flask by treatment with cell stripper (Cellgro; Manassas, Va.). Cells were counted, blocked and aliquots of 1.5×106 cells were incubated with 1.5 μg of anti-αVβ3 and anti-αVβ5 integrin antibodies (Millipore; Temecula, Calif.), 1.5 μg of mouse monoclonal antibody Desmoglein 2 (DSG2) (clone AH12.2, Santa Cruz Biotechnology Inc, Santa Cruz, Calif.) or untreated (control). Following primary antibody incubation, the cells were washed and incubated with Alexa fluor 488-conjugated goat anti-mouse immunoglobulin G (Invitrogen) diluted in PBS with 0.1% BSA. Cells were then diluted in PBS and a cytometric analysis of 10,000 events per sample was conducted using FACScan with CellQuest software (Becton Dickinson, Mountain View, Calif.).
5
Immunohistochemical Analysis of Cellular Markers
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After incubating with 10% FBS, the sections were incubated with primary antibodies overnight at 4°C, then, the primary antibodies were replaced by secondary antibody labels and cell nuclei were stained with DAPI. Primary antibodies: α-SMA (cat. no. Ab5694; dilution: 1:50; Abcam), CASP3 (cat. no. ab32351; dilution: 1:50; Abcam), IL-1β (cat. no. ab254360; dilution: 1:50; Abcam). Secondary antibodies: Alexa Fluor® 568-conjugated goat anti-rabbit immunoglobulin G (cat. no. A-11034; dilution, 1:250; Thermo Fisher Scientific), and Alexa Fluor® 488-conjugated goat anti-mouse immunoglobulin G (cat. no. A32727; dilution, 1:250; Thermo Fisher Scientific). Images were obtained by E2000U microscope (Nikon Corporation, Tokyo, Japan).
6
Immunofluorescence Staining of miMSCs
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miMSCs were fixed with 4% paraformaldehyde for 10 min. Following permeabilization with 0.2% Triton X-100 for 8 min, the cells were blocked with 5% FBS for 10 min and incubated sequentially for 90 min with primary antibody (mouse anti-preS2 monoclonal immunoglobulin G, 1:200, cat. no. sc-516176; Santa Cruz Biotechnology Inc., Dallas, TX, USA). Then, cells were incubated with secondary antibody (Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin G, 1:200, B40941; Thermo Fisher Scientific, Inc.) for 40 min. Between each step, cells were washed with PBS three times (3 min/time) and subsequently nuclear staining was performed using 4′,6-diamidino-2-phenylindole (1:10,000; Beyotime Institute of Biotechnology, Shanghai, China) for 5 min. Cells were examined with a fluorescence microscope (Olympus Corporation) at magnification, ×200. All steps were carried out at room temperature.
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