The cells were fixed in 4% paraformaldehyde for 30 min at room temperature, and then blocked with 10% goat serum (GTX27481, Gene-Tex, USA) for 1 h at room temperature. The samples were incubated at 4 °C overnight with the following primary antibodies, such as iNOS (1:100, ab15323, Abcam, UK), CD86(1:100, ab239075, Abcam, UK), Arg-1(1:100, ab239731, Abcam, UK), CD34(1:100, ab81289, Abcam, UK), α-SMA (1:100, ab124964, Abcam, UK), IBA(1:100, ab178846, Abcam, UK) and F4/80 (1:50, ab6640, Abcam, UK). Subsequently, the sections were incubated with mouse anti-rabbit IgG (1:1000, #4408/4409, Cell Signaling Technology, USA), rabbit anti-mouse IgG (1:1000, #4412/4413, Cell Signaling Technology, USA), and donkey anti-rat IgG (1:1000, A21209, Invitrogen, USA) for 1 h at room temperature. After washing, the sections were mounted with a DAPI-containing antifade solution and observed under a microscope (BX63, Olympus) and a confocal microscope (Zeiss, LSM800).
Gtx27481
Manufactured by GeneTex
Sourced in United States
GTX27481 is a high-performance electrophoresis system designed for efficient separation and analysis of nucleic acid and protein samples. It features a durable construction, adjustable voltage and current settings, and integrated cooling capabilities to ensure consistent and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Ab6640, by Abcam (2 mentions)
Lsm 800, by Zeiss (2 mentions)
Ab15323, by Abcam (2 mentions)
Ab124964, by Abcam (1 mentions)
Ab239731, by Abcam (1 mentions)
Ab239075, by Abcam (1 mentions)
Ab81289, by Abcam (1 mentions)
Ab178846, by Abcam (1 mentions)
Ab192029, by Abcam (1 mentions)
A21209, by Thermo Fisher Scientific (1 mentions)
Sc 6246, by Santa Cruz Biotechnology (1 mentions)
Ab13243, by Abcam (1 mentions)
Ab46545, by Abcam (1 mentions)
Bx41f, by Olympus (1 mentions)
Rabbit polyclonal anti α sma, by Abcam (1 mentions)
Alexa flour 488 goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
S36939, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti cd34, by Abcam (1 mentions)
4 protocols using gtx27481
1
Immunohistochemical Staining Protocol
2
Immunofluorescence Staining of BV-2 Cells
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The BV-2 cells were fixed in 4% paraformaldehyde for 30 min at room temperature and washed with PBS. Then the non-specific binding sites were blocked with 10% goat serum (GTX27481, Gene-Tex, USA) for 1 h at room temperature, and the samples were then incubated at 4 °C overnight with the following primary antibodies in PBS: iNOS (1:100, ab15323, Abcam, UK), Ym-1(1:100, ab192029, Abcam, UK), and F4/80 (1:50, ab6640, Abcam, UK). The following day, sections were incubated with mouse anti-rabbit IgG (1:1000, #4408/4409, Cell Signaling Technology, USA), rabbit anti-mouse IgG (1:1000, #4412/4413, Cell Signaling Technology, USA), and donkey anti-rat IgG (1:1000, A21209, Invitrogen, USA) for 1 h at room temperature. After rinsing, sections were mounted with a DAPI-containing antifade solution. Fluorescence signals were then observed under a microscope (BX63; Olympus) and a confocal microscope (Zeiss; LSM800).
3
Immunofluorescent Localization of LC3 and P21 in Mouse Heart Tissue
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Immunofluorescence staining was performed in order to determine the expression as well as the localization of LC3/P21 in the mice hearts. Briefly, for the heart section staining, all sections were autoclaved with citrate solution for antigen retrieval. After blocking with 10% goat serum (GTX27481; GeneTex, Sanantonio, TX, USA), the sections were incubated in LC3A/B (12741, CST) or P21 antibody (sc-6246, Santa Cruz Biotechnology, Dallas, TX, USA) overnight. Heart sections were subsequently incubated with secondary antibodies for 60 min at 37 °C, followed by visualization of the nuclei with DAPI. For immunohistochemistry, the tissue sections were heated for antigen retrieval, and then incubated with anti-heme oxygenase-1 (ab13243, Abcam, Cambridge, UK) or 4-hydroxynonenal (ab46545, Abcam), followed by incubation with goat anti-rabbit EnVisionTM+/HRP reagent, and finally staining with a DAB detection kit. The images were captured using a fluorescence microscope (OLYMPUS DX51, Tokyo, Japan) and then analysed using Image-Pro Plus software.
4
Quantifying Tumor Vasculature Markers
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The tumor sections (6 μm) were fixed in 4% paraformaldehyde for 3–4 h at room temperature, and rinsed with phosphate-buffered saline (PBS). After washing with PBS, the non-specific binding sites were blocked with 10% goat serum (GTX27481, GeneTex) for 1 h at room temperature. The samples were incubated with one or two primary antibodies, that is, mouse monoclonal anti-CD34 (1:50, Abcam, USA) and rabbit polyclonal anti-α-SMA (1:50, Abcam, USA), simultaneously in 1% goat serum at 4°C overnight. Sections were washed with PBS and incubated in the dark for 1 h with secondary antibodies: Alexa Flour® 568 goat anti-rabbit IgG (H+L) (1:200, A11011, Invitrogen) and Alexa Flour® 488 goat anti-mouse IgG (H+L) (1:200, A1106, Invitrogen). After washing three times with PBS for 5 min, the nuclei were stained with DAPI (S36939, Invitrogen) for 15 min, and the sections were then examined under a confocal scanning microscope (BX41F; Olympus, Tokyo, Japan). The ratio of α-SMA/CD34 was calculated by dividing the positive area of α-SMA adjacent to CD34-positive vessels by the total area of CD34-positive tumor vasculature under five 200× high-powered randomly chosen fields per slide.
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