Thunder imager tissue microscope

For O-GlcNac-Albumin immunofluorescence staining, samples dewax were pre-treated in PTLink Citrate Buffer pH 6. Then, sections were washed three times in TBS 0.1 M for 5 min each and incubated in blocking solution (2% donkey serum + 0.3% Triton X-100 + 0.25% BSA) in TBS 0.1 M for 60 min. Next, sections were incubated with the primary antibodies O-GlcNac (ab2739, Abcam) (1:200) and Albumin (ab8940, Abcam) (1:200) in blocking solution for 24 h at 4 °C. After incubation in the primary antibody, sections were rinsed with TBS 0.1 M three times for 5 min each and then incubated in the solution (0.3% Triton X-100 + 0.25% BSA in TBS 0.1 M) with the secondary antibodies: Alexa 488 anti-mouse (115-545-003, Jackson ImmunoResearch Labs) and Alexa 555 anti-Sheep (A21436, Thermo) 1:1000 for 60 min at room temperature. Sections were then washed and coverslipped with Fluorogel coverslip mounting solution. In all the histological staining, up to 4 representative microphotographs of each animal were taken with a Thunder Imager tissue microscope (Leica Microsystems) (immunofluorescence staining). Leica Las X 3.7.4 software was used for acquisition and analysis of immunofluorescence staining. Image J 1.52p software was used for the quantification of the staining area.