Fitc conjugated goat anti rabbit

Following drug treatment, the cells were fixed with 100% methanol for 6 min at −20°C, washed with PBS and stored at 4°C until further use. The cells were permeabilized through incubation in PBS containing 0.3% Triton X-100 and 5% goat serum for 30 min. A polyclonal antibody against phospho-EGFR was used at a 1:100 dilution, and a secondary antibody FITC-conjugated goat anti-rabbit (Invitrogen, Carlsbad, CA), was used at a 1:200 dilution. The first antibody was incubated overnight at 4°C and the second antibody was incubated for 2 hours at RT. The images were captured using an Olympus DP 71 camera (Tokyo, Japan) at 400× magnification.

After deparaffinization and rehydration, the sections were heated to 97 °C for 20 min in the presence of an antigen retrieval solution (CITRA, pH 6.0; Invitrogen, Carlsbad, CA) and cooled for 30 min. To block the endogenous peroxidase activity, all sections were incubated with hydrogen peroxide for 10 min and washed with PBS-Tween. The sections were then pre-incubated with 2% nonfat milk for 30 min at room temperature, followed by incubation with a mixture of two primary antibodies targeting estradiol and tryptase, respectively, overnight at 4 °C. After washing with PBS-Tween, the sections were incubated with a mixture of two fluorescent conjugated secondary antibodies (FITC conjugated Goat anti-Rabbit and Texas Red conjugated Goat anti-Mouse, Invitrogen, Carlsbad, CA) diluted in PBS, for 60 min at room temperature. Counterstaining was performed with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, Carlsbad, CA) for 20 min at room temperature. The sections were then washed in PBS-Tween and coverslipped using an anti-fade fluorescent mounting medium. After sealing with nail polish, the specimens were stored in the dark at 4 °C.

Gelatin-coated cover slips of serial tissue sections were incubated with anti-rabbit (1:100) IL-33 (Cloud-Clone Corp., Katy, TX) overnight at 4°C, followed by 3 five-minute washes with cold PBS with 0.1% Triton-X. The sections were then incubated with a FITC conjugated goat anti-rabbit (1:100, Invitrogen, Carlsbad, CA) at 37°C for one hour, followed by 3 five-minute washes with cold PBS with 0.1% Triton-X. Cover slips were then mounted on slides for visualization on a Zeiss fluorescent microscope (Zeiss, Thornwood, NY).

After the drug treatment, the cells were fixed with 100% methanol for 6 min at −20°C, then washed with PBS and left at 4°C until use. Cells were permeabilized by incubation in PBS containing 0.3% Triton X-100 and 5% goat serum for 30 min. A polyclonal antibody against phospho-cPLA₂ was used at a 1:100 dilution, and a secondary antibody FITC-conjugated goat anti-rabbit (Invitrogen, Carlsbad, CA), was used at a 1:200 dilution. The first antibody was incubated overnight at 4°C and the second antibody for 2 hours at RT. Images were captured with an Olympus DP 71 camera (Tokyo, Japan). The magnification level was 400 ×.