Rabbit anti bcl 2

Immunohistochemistry was performed on fresh‐frozen and paraffin‐embedded human brain sections.¹² Fresh‐frozen human brain sections were fixed in 4% PFA for 10 minutes at room temperature, followed by peroxidase enzyme deactivation. Human MS brain paraffin sections were de‐paraffinized and rehydrated followed by antigen retrieval (10‐mM citrate buffer, pH 6). After washing in PBS, peroxidase enzyme was deactivated using 3% H₂O₂ in 2% Triton X‐100 solution (in PBS) in both tissue types. Sections were blocked in 5% normal goat serum (0.3% Triton X‐100 in PBS) for an hour at room temperature, followed by overnight incubation in primary antibodies (rat anti‐proteolipid protein [PLP], 1:250, kindly provided by Dr. Wendy Macklin, University of Colorado; rabbit anti‐COL5A2—1:500, Boster Inc., rabbit anti‐RDX—1:200, Sigma; rabbit anti‐BCL2—1:200, Proteintech Inc.) at 4°C. The next morning, after washing in PBS sections were incubated in primary antibody host‐specific biotin‐tagged secondary antibody for 1 hour at room temperature, followed by Avidin‐Biotin complex staining as per the manufacturer's suggestion (Vector lab, cat#PK‐6100). Sections were washed in PBS, developed with 3,3′‐diaminobenzidine tablets (DAB, Sigma, cat#D5905) and 0.012% H₂O₂, dehydrated, and mounted before imaging.

Tissues were homogenized in RIPA plus buffer containing a cocktail of EDTA-free protease inhibitors. The homogenate was centrifuged at 12,000 rpm for 30 min at 4°C. Protein concentration was assayed using the BCA method, then loaded and subjected to electrophoresis in 10% SDS-PAGE gels, and transferred onto PVDF membranes. The membranes were then blocked in 5% BSA for 2 hour and then incubated with one of the following primary antibodies: rabbit anti-caspase-3 (1 : 500, Proteintech) or mouse anti-Bax (1 : 100, Proteintech) or rabbit anti-Bcl-2 (1 : 500, Proteintech) or mouse anti-Notch1(1 : 500, Santa Cruz) or mouse anti-Hes1 (1 : 1000, Santa Cruz), with gentle shaking at 4°C overnight. Then, horseradish peroxidase conjugated goat anti-mouse IgG (1 : 50000, Proteintech) or goat anti-rabbit IgG (1 : 50000, Proteintech) secondary antibodies were incubated with the membranes for 2 hours at room temperature. The immunopositive bands were visualized using an enhanced chemiluminescent substrate (Thermo Fisher) and Bio-Rad ChemiDoc XRS digital documentation system. The amount of protein expression is presented relative to the levels of β-actin.