The large intestines were removed and embedded in optimal cutting temperature (OCT) compound, snap-frozen using isopentane chilled in liquid nitrogen and kept at −80 °C until use. Cryosections were prepared using a cryostat Leica CM3054. For hematoxylin and eosin (H&E) staining, transversely oriented sections (10 μm) were cut at mid-point and stained as previously described [51 (link)]. The samples were digitally imaged using a Nikon Ti-E inverted fluorescence microscope equipped with a Lumenera Infinity Color CCD camera, and a Nikon Super Fluor 20× 0.75 NA objective lens (Nikon Inc., Melville, NY, USA). The digital images were processed using NIS-Elements AR version 4.30 (Nikon, Melville, NY).
Frozen tissue sections (10 μm) were fixed with 4% paraformaldehyde for 15 minutes at room temperature. After washing with PBS, the slides were blocked with 3% BSA for 1 hour. The slides were incubated with primary antibodies against p-STAT3 #9145, 1:1000, CST) at 4 °C overnight. After that, the slides were washed extensively with PBS and incubated with Alexa Fluor 488 (donkey anti-rabbit IgG, Invitrogen) for 1 hour at room temperature. The slides were sealed with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratory, Burlingame, CA). All images were taken with a Nikon Ti-E fluorescence microscope (magnification 20x) (Nikon, Melville, NY).
Frozen tissue sections (10 μm) were fixed with 4% paraformaldehyde for 15 minutes at room temperature. After washing with PBS, the slides were blocked with 3% BSA for 1 hour. The slides were incubated with primary antibodies against p-STAT3 #9145, 1:1000, CST) at 4 °C overnight. After that, the slides were washed extensively with PBS and incubated with Alexa Fluor 488 (donkey anti-rabbit IgG, Invitrogen) for 1 hour at room temperature. The slides were sealed with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratory, Burlingame, CA). All images were taken with a Nikon Ti-E fluorescence microscope (magnification 20x) (Nikon, Melville, NY).