Cebpd

Formalin‐fixed, paraffin‐embedded (FFPE) tissue sections were deparaffinized and rehydrated by using xylene and ethanol, respectively. After retrieval with citrate buffer at pH 6.0, the endogenous peroxidase activity of specimens was quenched by peroxidase‐blocking solution (Dako). The sections were incubated with the primary antibody against the target protein as indicated below: MYC (Abcam, ab32072), CEBPD (Abcam, ab184911), HK2 (Cell Signaling, 2867), FBXW7 (Abcam, ab105752), phospho‐AKT (Cell Signaling, 4060), phospho‐mTOR (Abcam, ab51044), phospho‐4E‐BP1 (Cell Signaling, 9644), phospho‐RPS6 (Abcam, ab80158), and MKI67 (Abcam, ab66155), followed by the incubation of a secondary antibody (REAL EnVision/HRP, rabbit/mouse [ENV], Dako). Staining was visualized with EAL DAB+ Chromogen diluted in REAL Substrate Buffer (Dako). Hematoxylin was used for the nuclear stain. Finally, the slices were dehydrated by soaking in various concentrations of ethanol and were mounted in UltraCruz Aqueous Mounting Medium with DAPI. The IHC staining results were examined with an optical microscope and quantified into H‐scores by three expert pathologists (Chien‐Feng Li, Tzu‐Ju Chen and Wan‐Shan Li ) as previously mentioned.²² Detailed information is shown in Supporting Information.

Frozen sections of whole brain (Control, ouabain, and ouabain + M8I groups) were mounted onto coated glass slides and the OCT embedding medium was dissolved in PBS at 23 °C. Subsequently, the sections were retrieved for 10 min at 90 °C and further blocked with a solution containing 10% normal donkey serum in PBS with 0.1% Tween 20 for 1 h at 23 °C. The sections were then incubated overnight at 4 °C with antibodies against GFAP (Abcam), CEBPD (Abcam), nitrotyrosine (Santa cruz), and Iba1 (Wako) in PBS. The slides were then washed thrice with TBST for 10 min each. After washing, sections were incubated with Alexa Fluor 488- or 555-conjugated secondary antibodies in PBS for 1 h. Following another three washes with TBST for 10 min each, the sections were cover-slipped using ProLong Gold antifade reagent with or without 4ʹ,6-diamidino-2-phenylindole (DAPI) at 23 °C for 10 min. The results were visualized using immunofluorescence confocal microscopy.