On day 19 after tumor injection, tumor tissues were collected from each group. The frozen sections were fixed in cold acetone for 15 minutes and incubated with rabbit anti‐mouse PD‐L1 (eBioscience) or rabbit anti‐mouse PD‐1 (eBioscience) + rat anti‐mouse CD8 (eBioscience) for 1 hour. Slides were washed with TBST for 5 minutes and secondary Abs (Alexa Fluor 647‐labeled goat anti‐rabbit Abs and/or Alexa Fluor 488‐labeled goat anti‐rat Abs; eBioscience) were added and incubated for 30 minutes. Slides were washed with TBST for 10 minutes and mounted with a Vectashield DAPI containing kit (Sigma, Saint Louis, MO, USA). PD‐1+CD8+ T cells and expression of PD‐L1 in the TME were then observed through a fluorescence microscope (Eclipse E800; Nikon, Tokyo, Japan).
Alexa fluor 488 labeled goat anti rat abs
Manufactured by Thermo Fisher Scientific
Alexa Fluor 488-labeled goat anti-rat Abs is a secondary antibody conjugate used in immunochemical applications. It is composed of goat-derived antibodies that specifically bind to rat primary antibodies, and are labeled with the fluorescent dye Alexa Fluor 488. This product can be used to detect and visualize target antigens in various immunoassay techniques.
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2 protocols using alexa fluor 488 labeled goat anti rat abs
1
Tumor Immune Cell Phenotyping
2
Immunofluorescent Analysis of CD11b+LOX-1+ Cells
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Tumor tissues were embedded in freeze embedding medium. The frozen sections were xed and incubated with anti-CD11b (eBioscience) and anti-LOX-1 antibodies (eBioscience) for 60 minutes. The secondary antibodies (Alexa Fluor 647-labeled goat anti-rabbit Abs and Alexa Fluor 488-labeled goat antirat Abs)(eBioscience) were then added and incubated for 30 minutes. The slides were mounted with a Vectashield DAPI-containing kit. Then CD11b + LOX-1 + cells in the tumor tissue were visualized with uorescence microscopy.
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