Immunohistofluorescence staining was used to evaluate the expression of dCK and CD31 in L1210-WT and L1210–10K tumors. Antibodies used in this study include: mouse anti-dCK (gift from Dr. Radu (31 (link))), rabbit mAb anti-CD31 (#77699, Cell Signal Technologies, Beverly, MA), Alexa Fluor 488-conjugated anti-rabbit IgG (#4412, Cell signaling Technologies), Alexa Fluor 594-conjugated anti-mouse IgG (#8890, Cell signaling Technologies). Tumors were embedded in optimal cutting temperature compound (OCT), frozen at −80 °C and cryosectioned at the thickness of 5 μm. Tumor sections were fixed and permeabilized using cold acetone, followed by blocking using 1% bovine serum albumin (BSA in PBS) at room temperature for 1h. Primary and secondary antibodies were applied to the tissue sequentially using recommended dilutions, with extensive washing with PBS in between. DAPI was used to stain the nucleus. Cover glass was mounted using the Prolong Antifade Mountant (Thermo Fisher Scientific). Tumor sections were imaged using a confocal laser scanning microsope (Olympus FV3000, Tokyo, Japan).
Alexa fluor 594 conjugated anti mouse igg
Manufactured by Cell Signaling Technology
Alexa Fluor 594-conjugated anti-mouse IgG is a fluorescently labeled secondary antibody that binds to mouse immunoglobulin G (IgG). It is designed for use in various immunodetection techniques, such as immunofluorescence, flow cytometry, and Western blotting.
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2 protocols using alexa fluor 594 conjugated anti mouse igg
1
Immunofluorescence Analysis of dCK and CD31
2
Immunofluorescence Staining of HUVEC-T1 Cells
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PUMC-HUVEC-T1 cells were cultured on coverslips in 24-well plates. Once the cells reached approximately 50% confluence, immunofluorescence staining was conducted following a previously described method [5 (link)]. The coverslips were incubated overnight at 4 °C with mouse anti-CD31 (1:500, 66065-2-Ig, Proteintech, Wuhan, China) and rabbit anti-CXCR2 (1:50, 20634-1-AP, Proteintech) or anti-NF-κB p65 (1:500, #8242, Cell Signaling Technology, Danvers, MA, USA) diluted in blocking buffer. Following incubation, the coverslips were washed and incubated with secondary Alexa Fluor 594 conjugated anti-mouse IgG and Alexa Fluor 488 conjugated anti-rabbit IgG (1:1000, #8890, and #4412, Cell Signaling Technology) for 1 h at room temperature. After washing, the nuclei were counterstained using a mounting medium containing DAPI. Fluorescent images were captured using a wide-field microscope (IX81, Olympus, Tokyo, Japan).
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