After 48 h of NPs injection and laser irradiation, the tumors were removed and fixed in 4% paraformaldehyde for 24 h, paraffin-embedded, and sectioned into 10–20 µm thickness. Next, the sections were dewaxed in xylene and rehydrated in a graded series of alcohol. Antigen retrieval was conducted in citric acid buffer (pH 6.0) for 10 min at 98 °C . The tumor sections were blocked in 2% normal goat serum (1:200; Protein Tech Group, Chicago, USA) for 1 h at room temperature. Then, the sections were incubated overnight at 4 °C with an anti-cluster of differentiation (CD)-31 antibody (1:500; ab28364; Abcam, Cambridge, UK) and α-smooth muscle actin (SMA) antibody (1:100; 14,395-1-AP; Protein Tech Group, Chicago, USA). Next, these sections were washed and incubated with rhodamine-conjugated goat anti-rabbit IgG-FITC (1:200; sc-2359; Santa Cruz Biotechnology, Dallas, USA) for 40 min at room temperature. The nucleus was counterstained using DAPI for 15 min at room temperature (00-4959-52; Invitrogen; Thermo Fisher Scientific, Massachusetts, USA), and the tissues were visualized using a fluorescent microscope (× 20–× 1000; Leica DM6000B; Leica Microsystems GmbH, Wetzlar, Germany). For each section, a total of 5 images were taken from randomly selected fields of view.
Normal goat serum (ngs)
Manufactured by Proteintech
Sourced in China, United States
Normal goat serum is a biological reagent derived from the blood of healthy goats. It is commonly used in various immunological and cell culture applications as a blocking or diluent solution to minimize non-specific binding in immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Ab28364, by Abcam (2 mentions)
Sc 2359, by Santa Cruz Biotechnology (2 mentions)
Dm6000b, by Leica (2 mentions)
Chamber slide, by BD (2 mentions)
Ab2349, by Abcam (1 mentions)
Epr20164 278, by Abcam (1 mentions)
Alexa fluor 594 goat anti mouse immunoglobulin g, by Proteintech (1 mentions)
Image analysis system, by Zeiss (1 mentions)
Tubulin tracker red, by Beyotime (1 mentions)
Anti sirt1 antibody, by Proteintech (1 mentions)
Anti foxo1 antibody, by Cell Signaling Technology (1 mentions)
Chamber slide, by Merck Group (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Fhl2 antibody, by Proteintech (1 mentions)
5 protocols using normal goat serum (ngs)
1
Immunohistochemical Analysis of Tumor Vasculature
2
Immunohistochemical Analysis of Tumor Vasculature
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The mice were sacrificed and tumors were harvested. The tumor tissues (10–20 µm thick) were fixed in 4% paraformaldehyde for 24 h at 4°C, paraffin-embedded and sectioned. Following on, the tissues were dewaxed in Xylene and rehydrated with graded alcohol. Antigen retrieval was conducted in citric acid buffer (PH 6.0) for 10 min at 98°C. Tumor sections were blocked in 2% normal goat serum (1:200; ProteinTech Group, Inc., Chicago, IL, USA) for 1 h at room temperature. Then, the sections were incubated overnight at 4°C with an anti-cluster of differentiation (CD)-31 antibody (1:500; ab28364; Abcam, Cambridge, UK), an α-smooth muscle actin (SMA) antibody (1:100; 14395-1-AP; ProteinTech Group, Inc.), an anti-VEGFR2 antibody (1:100; ab2349; Abcam) and an anti-AGR2 antibody (1:500; EPR20164-278; Abcam). Then those sections were washed and incubated with rhodamine-conjugated goat anti-rabbit IgG-FITC (1:200; sc-2359; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 40 min at room temperature. The nuclei were counterstained using DAPI for 15 min at room temperature (00-4959-52; Invitrogen; Thermo Fisher Scientific, Inc.), and the tissues were visualized using a fluorescent microscope (×20 to ×1,000; Leica DM6000B; Leica Microsystems GmbH, Wetzlar, Germany) at magnification ×20. For each section, a total of 8 images were taken of randomly selected fields of view.
3
Immunofluorescence Visualization of ATF4
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On the third day of culture, cells in a chamber slide (BD Biosciences, USA) were fixed in 4% paraformaldehyde and then permeabilized with 0.4% Triton X-100. After washing, the cells were blocked with normal goat serum (Proteintech, China) for 1 h and then incubated with ATF4 (1:200, RRID: AB_205875, Santa Cruz, USA) overnight at 4°C. After washing with phosphate-buffered saline, Alexa Fluor 594 goat anti-mouse immunoglobulin G (red, 1:200, Proteintech, China) was used as secondary antibodies, and the cells were incubated in the dark for 2 h. The nuclei were counterstained with 4′, 6′-diamidino-2-phenylindole (blue). Staining intensity was examined using a fluorescence microscope.
4
Immunofluorescence Analysis of Stem Cell Markers
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The isolated primary ESCs in the chamber slide (BD Biosciences) were fixed with 4% paraformaldehyde and then permeabilized with 0.4% Triton X-100. After washing, the cells were blocked with normal goat serum (Proteintech, China) for 1 h and then incubated with anti-SIRT1 antibody (1:100, 3 μg/ml, Proteintech) or anti-FOXO1 antibody (1:100, 0.88 μg/ml, Cell Signaling Technology, Danvers, MA, United States) or without primary antibodies as the negative control overnight at 4°C. After washing with phosphate-buffered saline (PBS), the cells were incubated in darkness for 2 h at room temperature with Alexa Fluor 488 (green color)- and 598 (red color)-labeled secondary antibodies (Proteintech). Nuclei were stained with 4′,6′-diamino-2-phenylindole (DAPI) (1 μg/ml).
Immunofluorescence staining of the cytoskeleton was performed according to the protocols of Tubulin-Tracker Red (Beyotime Biotechnology, Shanghai, China). After primary ESC was fixed as mentioned above, 100 μl liquid of Tubulin-Tracker Red was added and incubated at room temperature in dark for 40 min. Then PBS containing 0.1%Triton X-100 was used for washing for three times. Nuclei were stained with 4′,6′-diamino-2-phenylindole (DAPI) (1 μg/ml).
All images were obtained with a fluorescence microscope and camera connected to a computer with an image analysis system (Zeiss).
Immunofluorescence staining of the cytoskeleton was performed according to the protocols of Tubulin-Tracker Red (Beyotime Biotechnology, Shanghai, China). After primary ESC was fixed as mentioned above, 100 μl liquid of Tubulin-Tracker Red was added and incubated at room temperature in dark for 40 min. Then PBS containing 0.1%Triton X-100 was used for washing for three times. Nuclei were stained with 4′,6′-diamino-2-phenylindole (DAPI) (1 μg/ml).
All images were obtained with a fluorescence microscope and camera connected to a computer with an image analysis system (Zeiss).
5
Immunofluorescence Analysis of FHL2 in KGN and hGCs
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After treatment with FSK for 4 h, KGN cells were fixed in a chamber slide (Millipore, Billerica, USA) in 4% paraformaldehyde and then permeabilised with 0·4% Triton X-100. After washing, the cells were blocked with normal goat serum (Proteintech, Wuhan, China) for 1 h and then incubated with FHL2 antibody (1:50; Proteintech) overnight at 4 °C. After washing, Alexa Fluor 594-conjugated anti-mouse IgG (green) (1:200; Proteintech) was used as the secondary antibody, and the cells were incubated in the dark for 2 h. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Cells were observed and imaged using a fluorescence microscope (Zeiss, Oberkochen, Germany).
Three days after plating, same method of immunofluorescence staining was used in hGCs after treatment with hCG (10 IU/ml, 4 h).
Three days after plating, same method of immunofluorescence staining was used in hGCs after treatment with hCG (10 IU/ml, 4 h).
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