Non fat dry milk

Whole cell extracts were prepared using RIPA buffer (Thermo Fisher) supplemented with protease inhibitors (cOmplete; Roche; Indianapolis, IN). Protein concentration was measured by bicinchoninic acid (BCA assay; Thermo Fisher). Total protein (30 μg) was electrophoresed in 4–20% Bis/Tris gels (Genscript; Piscataway, NJ). Separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) and incubated with indicated primary or secondary antibodies in blotting buffer containing 1X phosphate-buffered saline (PBS), 0.2% Tween-20, and 10% (w/v) non-fat dry milk (Thermo Fisher). The enhanced chemiluminescence detection system (Thermo Fisher) was used to detect proteins as previously described^{19 (link)}.

7-week old male C57BL/6 or FVBN wild type mice were sacrificed after 5-days following pilocarpine injection and induced status epilepticus (see above). The mice were anesthetized with isoflurane 4% and perfused with cold 4% PFA (pH 7.4). The brains were surgically removed and fixed in cold 4% PFA for at least four hours or overnight at 4°C. The fixed brains were cryoprotected in 30% Sucrose overnight at 4°C. Hippocampal coronal sections (12 μm) were cut using a Leica Biosystem cryostat. The coronal sections were stored at -20°C for later immunostaining. Before immunostaining, the sections were pretreated with 3% hydrogen peroxide in methanol for 5 min and washed with PBS for another 5 min. Thereafter, the coronal sections were incubated with primary antibodies 1:100, as listed in Supplemental Material, diluted in 10% donkey serum (Abcam), 5% Non-fat dry milk, 4% BSA (Thermo Fisher), and 0.1% Trion X-100 (Sigma Aldrich) overnight at 4°C in moisturized chamber. The next day, the sections were washed with PBS for 5 minutes three times and incubated with secondary antibody 1:150; as listed in Supplemental Material, diluted in donkey blocking buffer at room temperature for one hour. Afterwards, the sections washed three times with PBS for 5 minutes and then mounted with flourished mounted DAPI (Abcam) for confocal imaging.

After three washes with PBS, cells were suspended in 4 °C cell lysis buffer (Bio-Rad, CA, USA). Proteins in the lysates were quantified to equivalent and separated by 12% SDS-PAGE. They were then transferred to nitrocellulose membranes (Bio-Rad, CA, USA). Membranes were blocked with 5% non-fat dry milk (Yili, China) in Tris-buffered saline Tween-20 buffer (Thermo Fisher Scientific, MA, USA). After blocking, membranes were incubated with primary antibodies at 4 °C overnight. For western blotting primary antibodies, anti-Sox-9, anti-p53, and anti-JNK were purchased from Cell Signaling Technology (MA, USA). Anti-aggrecan was purchased from Santa Cruz Biotechnology (TX, USA). Anti-collagen-X and anti-collagen-II were purchased from Abcam (CA, USA). After primary antibody incubation, membranes were fully washed and incubated with secondary antibodies (Beyotime, China) at room temperature for 1 h. Finally, membranes were visualized with ECL Prime (Thermo Scientific, CA, USA). GAPDH expression level was used as an internal control.

Western blot analysis to confirm the full-length ITIH5, ITIH5^681aa and ITIH5^161aa was performed as recently described [8 (link)], with slight modifications for the truncated polypeptides. After blocking in Tris-buffered saline containing 0.01% Tween-20 (TBS-T) and 5% non-fat dry milk (Roth, Karlsruhe, Germany), blots were probed with the primary antibody Penta-His-tag (C-Term) ((P-21315); 1:1000, Invitrogen, Carlsbad, CA, USA) in a blocking solution buffer, washed and incubated with goat anti-mouse 1:5000 ((P0447); Dako, Glostrup, Dänemark) secondary peroxidase-conjugated antibodies. Antibody detection was accomplished with Pierce ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL, USA). Equal protein loading was monitored by using a β-actin-specific antibody.