Gtx110520

Cells were first lysed with RIPA lysis buffer to collect cellular proteins. Then, the protein was separated by 10% SDS‐PAGE and then electro‐transferred to PVDF membrane. The protein on the membrane was detected with a suitable primary antibody. Finally, the protein was incubated with the secondary antibody for 1 h, and the protein signal was detected by the luminometer ECL. Primary antibodies were as follows: rabbit anti‐INOS (1:1000, GTX130246, GeneTex), mouse anti‐β‐actin (1:5000, RAB0100, Frdbio), rabbit anti‐IL‐1β (1:1000, YT2322, Immunoway), mouse anti‐PKM2 (1:1000, 60268‐1‐Ig, Proteintech), Mouse anti‐Histone H3.1(1:1000, GB11102, Servicebio), rabbit anti‐TNF‐α (1:1500, GTX110520, GeneTex), rabbit anti‐ALPK1 (1:6000, Abcam) and mouse anti‐GAPDH (1:5000, RAB0101, Frdbio).

After overnight fixation in 4% PFA, the brains were processed for paraffin embedding, and once included, serial sagittal 4 μm thickness sections were cut using a microtome (Microm HM 340E). The slides were dewaxed using xylol and hydrated in decreasing concentration of ethanol until PBS 0.1 M, and after hydration, the immunofluorescence assay was performed as follows. Briefly, brain sections containing the hippocampus were subjected to heat-induced epitope retrieval and blocked with 10% BSA for 90 minutes at room temperature. Then, samples were immunolabeled at 4°C overnight using the following primary antibodies at 1 : 100 dilution: anti-Iba1 (GTX101495, Genetex), anti-CD68 (GTX37743, Genetex), and anti-TNFα (GTX110520, Genetex). The next day, the slides were washed and incubated with the secondary antibody (SAB4600310, Sigma Aldrich Co.) diluted at 1 : 500 during 3 hours at room temperature. Then, sections were washed repeatedly and covered with Vectashield medium (Vector Laboratories, Burlingame CA) containing DAPI to counterstain nuclei.

We previously found hippocampal shrinkage and inflammation in KA-epilepsy models, as shown with hematoxylin and eosin and GFAP stains, respectively [16 ]. Both abnormalities improved with sonication using 0.25-MI and I_SPTA 0.5 W/cm² parameters without thermal damage (Supplementary S2) [16 ].
Based on the neuroprotective hypothesis that might link to FUS-assisted anti-inflammatory effect [14 (link), 16 ], we further examined the expressions of the ionized calcium-binding adaptor molecule 1 (IBA-1) and TNF-α [26 (link), 27 (link)] after two FUS treatments. Non-KA, KA only, and KA + FUS animals were anesthetized with isoflurane and sacrificed by transcardial perfusion with 0.9% saline at week 20. Brains were removed and fixed in 10% buffered neutral formalin. Fixed brains were dehydrated by gradient osmosis in 10%, 20%, and 30% sucrose solutions at 4℃. After dehydration, the brains were cut into a series of coronal blocks and embedded in optimal cutting temperature compound and were serially sectioned at 20 μm for IBA-1 (GeneTex, GTX635399, 1:500) and TNF-α (GeneTex, GTX110520, 1:500) of IHC staining. Histological changes in hippocampus and striatum were evaluated after two I_SPTA 0.5 W/cm² FUS treatments. The relative change of positive signals in IHC staining was quantified while using the contralateral untreated site as the baseline (via Image J [25 ]).

Large proteins were isolated from mouse cells using a radioimmunoprecipitation assay (RIPA) buffer. Western blotting was then carried out. The main primary antibodies included TNF-α (1:1000 dilution, GTX110520; GeneTex, San Antonio, TX, USA), vimentin (1:4000 dilution, ab92547, Abcam, Cambridge, UK), α-SMA (ab5694; Abcam, Cambridge, UK), collagen 1 (COL1, 1:4000 dilution, ab34710, Abcam, Cambridge, UK), and β-actin (AC026; ABclonal, Wuhan, China). They were then incubated with goat anti-rabbit or anti-mouse secondary antibodies (074–1506/074-1806, Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA). Immunoblot target bands were visualized using ECL Prime Western Blotting Detection Reagent (ZD310A, ZomanBio, Beijing, China). β-actin was used as an internal reference.