The expression of CD44, P63 and ABCG2 markers (as stemness markers of LESCs), was evaluated using flow cytometry. The cultured LESCs were harvested and re-suspended in PBS. Then the cells were rinsed with cold wash buffer (PBs containing 2% FBS and 0.1% sodium azide (Merck). Cells that were to be marked with surface antibodies (CD44 and ABCG2) were incubated with PE Mouse Anti-Human CD44 and APC-conjugated mouse anti-human ABCG2 (BD Pharmingen Inc) for 30 min at room temperature in dark. Cell staining with isotype-matched irrelevant antibodies (BD Pharmingen) was also performed. For intracellular staining cells were fixed with 1% cell fix for 5 minutes at 4°C and washed with 1 mL of ice-cold PBS. Then 0.2% saponin was added and incubated for 10 minutes. Then cells were centrifuged and incubated on ice with rabbit anti-human P63-α primary antibody for 1 h. Afterward, cells were washed twice with PBS and incubated with 10 μL FITC-conjugated goat anti-rabbit (Santa Cruz) secondary antibody for 30 minutes at room temperature in dark. Then cells were rinsed and analyzed with FACS CALIBUR flow cytometer (Becton Dickinson, Franklin Lakes, USA). Final data were analyzed by version 7.6 of FlowJo® software. Unstained controls were used as negative controls in the histograms to detect the percentage of positive cells.
Fitc conjugated goat anti rabbit
Manufactured by Santa Cruz Biotechnology
Sourced in United States
FITC-conjugated goat anti-rabbit is a secondary antibody that is conjugated to the fluorescent dye fluorescein isothiocyanate (FITC). It is designed to bind to and detect primary rabbit antibodies in various immunoassays and imaging applications.
Automatically generated - may contain errors
2 protocols using fitc conjugated goat anti rabbit
1
Evaluation of Stemness Markers in LESCs
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The expression of CD44, P63 and ABCG2 markers (as stemness markers of LESCs), was evaluated using flow cytometry. The cultured LESCs were harvested and re-suspended in PBS. Then the cells were rinsed with cold wash buffer (PBs containing 2% FBS and 0.1% sodium azide (Merck). Cells that were to be marked with surface antibodies (CD44 and ABCG2) were incubated with PE Mouse Anti-Human CD44 and APC-conjugated mouse anti-human ABCG2 (BD Pharmingen Inc) for 30 min at room temperature in dark. Cell staining with isotype-matched irrelevant antibodies (BD Pharmingen) was also performed. For intracellular staining cells were fixed with 1% cell fix for 5 minutes at 4°C and washed with 1 mL of ice-cold PBS. Then 0.2% saponin was added and incubated for 10 minutes. Then cells were centrifuged and incubated on ice with rabbit anti-human P63-α primary antibody for 1 h. Afterward, cells were washed twice with PBS and incubated with 10 μL FITC-conjugated goat anti-rabbit (Santa Cruz) secondary antibody for 30 minutes at room temperature in dark. Then cells were rinsed and analyzed with FACS CALIBUR flow cytometer (Becton Dickinson, Franklin Lakes, USA). Final data were analyzed by version 7.6 of FlowJo® software. Unstained controls were used as negative controls in the histograms to detect the percentage of positive cells.
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The expression of CD44, P63 and ABCG2 markers (as stemness markers of LESCs), was evaluated using flow cytometry. The cultured LESCs were harvested and re-suspended in PBS. Then the cells were rinsed with cold wash buffer (PBs containing 2% FBS and 0.1% sodium azide (Merck). Cells that were to be marked with surface antibodies (CD44 and ABCG2) were incubated with PE Mouse Anti-Human CD44 and APC-conjugated mouse anti-human ABCG2 (BD Pharmingen Inc) for 30 min at room temperature in dark. Cell staining with isotype-matched irrelevant antibodies (BD Pharmingen) was also performed. For intracellular staining cells were fixed with 1% cell fix for 5 minutes at 4°C and washed with 1 mL of ice-cold PBS. Then 0.2% saponin was added and incubated for 10 minutes. Then cells were centrifuged and incubated on ice with rabbit anti-human P63-α primary antibody for 1 h. Afterward, cells were washed twice with PBS and incubated with 10 μL FITC-conjugated goat anti-rabbit (Santa Cruz) secondary antibody for 30 minutes at room temperature in dark. Then cells were rinsed and analyzed with FACS CALIBUR flow cytometer (Becton Dickinson, Franklin Lakes, USA). Final data were analyzed by version 7.6 of FlowJo® software. Unstained controls were used as negative controls in the histograms to detect the percentage of positive cells.
2
Immunohistochemical Analysis of VEGF, Slit2, and Robo1
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Human fibrovascular membrane tissues were snap-frozen and 6 μm sections were cut. Thawed tissue sections were air dried, placed in 4% PFA for 20 min for fixing, washed with PBS, and blocked with 10% normal goat serum for 1 h at 37°C. Next, 1:100 anti- VEGF antibody (Santa Cruz, Santa Cruz, CA) in combination with either 1:200 anti-Slit2 polyclonal antibody (Millipore, Temecula, CA) or 1:100 anti-Robo1 polyclonal antibody (Abcam, Cambridge, UK) together at the same time were applied to the tissue sections at 4°C overnight and incubated for 1 h at 37°C with 1:100 fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse secondary antibodies (Santa Cruz, CA, USA) as appropriate. Following incubation, the slides were washed, and cell nuclei were stained with 4’, 6’-diamino-2- phenylindole (DAPI). Images were acquired using a fluorescence microscope equipped with a digital camera. For each of the immunostaining procedures, negative controls included the omission of the primary antibody and the use of an irrelevant polyclonal or isotype-matched monoclonal primary antibody. In all cases, negative controls showed only faint, insignificant staining.
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