Anti sk2

The BLA was prepared for western blotting analysis using Syn‐PER Synaptic Protein Extraction Reagent (Thermo Fisher, USA), resulting in both a functional synaptosome fraction and a cytosolic fraction.
²¹ (link) The protein concentration was determined by using BCA. SDS–PAGE gels were used to separate the same amounts of proteins before transfer to PVDF membranes. The PVDF membrane was blocked with 5% skim milk or 3% BSA (bovine serum albumin) at room temperature for 2 h, and then incubated overnight at 4°C with anti‐SK2 (1:500; Alomone labs, Israel), anti‐PKA (1:1000 Abcam ab38949), anti‐pPKA (phospho S96, 1:1000 Abcam ab32390), anti‐MPP2 (1:1000; Abcam, USA), anti‐GAPDH (1:1000), or anti‐β actin (1:3000) primary antibodies. After washing with TBST, the PVDF membrane was incubated with an HRP‐conjugated secondary antibody (1:1000) for 45 min at room temperature. Protein bands were illuminated using the BeyoECL Moon kit and captured using the Image ProPlus image analysis system (Media Cybernetics, Inc., Rockville, MD, United States).

Male mice were deeply anesthetized followed by serial intracardial perfusions with 0.9% saline (100 mL/100 g), and 4% polyformaldehyde in phosphate buffer (100 mL/100 g). Whole brains were rapidly dissected and postfixed with 4% polyformaldehyde at 4°C overnight and cryoprotected with 30% sucrose at 4°C. Then, the brain tissues were embedded, frozen in OCT at −20°C, and sectioned into 30‐μm slices using a Leica microtome. Brain sections were permeabilized with 0.1% Triton X‐100 in PBS, blocked with 5% normal bovine serum in PBS, and incubated with primary antibodies anti‐CaMKII‐ɑ (1:1000, Invitrogen, USA), anti‐c‐Fos (1:1000, Abcam, USA) or anti‐SK2 (1:100, Alomone labs, Israel) at 4°C overnight. Subsequently, the brain slices were rinsed three times, incubated with an Alexa Fluor 488‐conjugated anti‐rabbit (1:1000, Invitrogen, USA) or Alexa Fluor 594‐conjugated anti‐mouse antibody (1:1000, Invitrogen, USA) at r/t for 2 h, and mounted on positively charged slides. Images were obtained with a confocal laser scanning microscope.