Ab16659

Brains were collected and fixed overnight with 4% PFA in PBS at 4°C and dehydrated in 30% sucrose in PBS at 4°C. For sections or isolated cells, cryo‐sections (40 μm thick) or coverslips were fixed with 4% PFA/PBS, and permeabilized with 0.5% Triton X‐100 for 10 min and blocked in blocking buffer (0.3% Triton X‐100, 2% Bovine Serum Albumin) for 1 h at room temperature. Primary antibodies were incubated at 4°C overnight. The primary antibodies used in this study are as follows: rabbit anti‐Arid1a (1:1,000, HPA005456, Sigma), pig anti‐Vgult1 (1:1,000, ab5905, Millipore), rabbit anti‐H3K27ac (1:1,000, ab4729, Abcam), chick anti‐MAP2 (1:1,000, Cat No.822501, Biolegend), rabbit anti‐PSD95(1:1,000, ab16659, Abcam), mouse anti‐synaptophysin (1:1,000, ab13552, Abcam), mouse anti‐Oct‐3/4 (1;1,000, Cat No. sc‐5,279, Santa Cruz), rabbit anti‐Nanog (1:1,000, Cat No. 14295–1, Proteintech), and rat anti‐BrdU (1:1,000, Cat No. ab6326, Abcam). Sections or coverslips were washed in PBS and incubated with secondary Alexa Fluor‐conjugated antibodies (Invitrogen) for 1.5 h at room temperature, washed three times in PBS, and mounted with an adhesion antifade medium. We used a confocal laser‐scanning microscope to obtain images and ImageJ to analyze the results.

Western blot analysis was performed as previously published (Liu et al, 2017 (link)). The brain tissues were lysed in cold RIPA buffer (Beyotime, P0013B) containing a protease inhibitor cocktail (Roche). After centrifugation, the supernatants of the sample's concentration were measured using a BCA protein assay kit (Biomed, PP0102). Protein samples were loaded in 8–15% SDS‐PAGE gels (Bio‐Rad) and transferred into PVDF membranes (Millipore). The membrane was blocked with 5% milk in TBST (TBS + 0.05% Tween‐20) for 1 h at room temperature and incubated at 4°C overnight with primary antibodies. The following antibodies were used: anti‐tubulin (HRP Conjugated; 1:5,000, BE3312, EASYBIO), mouse anti‐β‐actin (1:10,000, A5441, Sigma), rabbit anti‐Arid1a (1:2,000, HPA005456, Sigma), rabbit anti‐H3K27ac (1:1,000, ab4729, Abcam), rabbit anti‐PSD95(1:1,000, ab16659, Abcam), mouse anti‐Oct‐3/4 (1;1,000, Cat No. sc‐5,279, Santa Cruz), rabbit anti‐Nanog (1:1,000, Cat No. 14295–1, Proteintech), rabbit recombinant anti‐Glutamate Receptor 1 (AMPA subtype) antibodies (1:1,000, ab109450, Abcam), and rabbit recombinant anti‐CaMKII antibodies (1:1,000, ab52476, Abcam). Secondary antibodies conjugated to HRP were incubated at room temperature for 1 h. Protein bands were visualized by enhanced chemiluminescence reagent (ECL, Pierce) and quantified using ImageJ.

The formalin-fixed tumours were dehydrated, embedded in paraffin, and sliced into sections of 4 μm, according to standard procedures. To verify the MTC origin of the tumours, sections from the mock-treated control group were stained with haematoxylin and eosin (H&E) for morphological examination, and by using antibodies for MTC markers chromogranin A (CgA, dilution 1:500; ab68271, Abcam, Cambridge, England), synaptophysin (Syn, dilution 1:25; ab16659, Abcam), and calcitonin (Ctn, dilution 1:1000; A0576, Dako, Glostrup, Denmark). Antibodies were incubated for 1 hour.
For the immunohistochemical (IHC) staining, tumour sections were collected on glass slides and then treated with EnVision^™ FLEX Target Retrieval Solution (high pH) using a PT-Link (Dako). The staining was done in an Autostainer Link using EnVision^™ FLEX (Dako) according to the manufacturer’s instructions. Positive and negative controls were included in each run. A microscope (20x magnification, Eclipse E1000, Nikon Instruments, Amsterdam, Netherlands) equipped with a camera (ProgRes C7, Jenoptik, Jena, Germany) was used for imaging of the stained tumour sections.