MMP-2 Antibody (2C1): sc-13594

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:500) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Protocol tips
-Mouse (1:500) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Publication protocol

Brain samples were extracted from the ipsilateral cerebral cortex with radioimmunoprecipitation buffer (Beyotime Institute of Biotechnology, Haimen, China) and centrifuged to remove the insoluble material (12,000 × g for 15 min at 4°C). Protein concentrations were determined using a BCA protein assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. A total of 30 µg protein were separated on 10% SDS-PAGE gels. Protein bands were then transferred to polyvinylidene difluoride membranes and incubated for 2 h at 37°C in Tris-buffered saline plus 0.1% Tween 20 (TBST) containing 5% skim milk. Membranes were incubated overnight at 4°C with primary antibodies against VEGF (1:1,000; catalog no. ab1316; Abcam), MMP-2 (1:500; catalog no. sc-13594; Santa Cruz Biotechnology, Inc.), MMP-9 (1:1,000; catalog no. ab76003; Abcam), occludin (1:500; catalog no. sc-271842; Santa Cruz Biotechnology, Inc.) and collagen-IV (1:500; catalog no. sc-11360; Santa Cruz Biotechnology, Inc.). The membranes were then incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (1:500; catalog nos. ZDR-5306 and ZDR-5307; ZSGB-Bio, Beijing, China) for 1 h at room temperature after washing the membranes three times with TBST. β-actin (1:500; catalog no. TA-09; ZSGB-Bio) expression was determined as a loading control. Labeled proteins were visualized by chemiluminescence using an enhanced chemiluminescence kit (Beyotime Institute of Biotechnology). The intensity of the bands was measured using the ChemiDoc detection system and Quantity One software version 4.6.8 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

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Manufacturer protocol

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