Immunocytochemical Analysis of Sglt1 in Mouse Uterus

The uterus was removed and immediately submerged into the fixative (4% p-formaldehyde) and further processed as described previously for mouse organs³⁸ . Immunocytochemistry was performed on 4-µm thick tissue cryosections described previously^{38 ,39} . Following antigen retrieval and washing steps in the detergent-containing buffers, cryosections were incubated for 30 min in 1% w/v bovine serum albumin solution (in PBS) to block nonspecific binding of the antibodies, then incubated with a previously described^{40 (link)} non-commercial, affinity purified, rabbit-raised, anti-mouse Sglt1 antibody (mSglt1-Ab; 1:100 in PBS) at 4 °C overnight. The samples were then rinsed, incubated in secondary GARCY3 antibody (1.6 μg/ml in PBS;CY3-labeled goat anti-rabbit IgG; # 111-165-003, Jackson ImmunoResearch Laboratories, USA) at room temperature for one hour, rinsed, overlayed with fluorescence fading retardant Vectashield (Vector Laboratories, USA). The staining was examined under the fluorescence microscope (Opton III RS; Opton Feintechnik, Germany). The images were captured using Spot RT Slider camera and software (Diagnostic Instruments, USA) and imported and processed in Adobe Photoshop 6.0.