Immunofluorescence Analysis of Cerebral Ischemia

At 24 hours following ICH, mice were perfused under deep anesthesia with cold phosphate-buffered saline (PBS, pH 7.4), then infused with 10% formalin. Brains were removed and fixed in formalin at 4°C for a minimum of 3 days. Samples were dehydrated with 30% sucrose in phosphate-buffered saline (PBS, pH 7.4) and the frozen coronal slices (10 μm thick) were sectioned in cryostat (CM3050S; Leica Microsystems). Immunofluorescence was performed as previously described.^{22 (link),36 (link)} Anti-PDGFR-β antibody (1:100, Santa Cruz), anti-p-p38 MAPK (1:100, Santa Cruz), anti-p-MK2 (1:100, Santa Cruz) anti-ICAM-1(1:100, Santa Cruz), anti-SMemb (1:100, abcam) were incubated separately with primary antibodies: anti-smooth muscle actin (1:1000, Santa Cruz) overnight at 4°C. It was then incubated with the appropriate fluorescence conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA). The slices were observed underneath a fluorescence microscope (Olympus BX51, Olympus Optical Co. Ltd, Japan), and pictures were taken with software MagnaFire SP 2.1B (Olympus, Melville, NY).

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Yang P., Wu J., Miao L., Manaenko A., Matei N., Zhang Y., Xu L., Pearce W.J., Hartman R.E., Obenaus A., Zhang J.H., Xu F, & Tang J. (2016). Platelet-derived growth factor receptor-β regulates vascular smooth muscle cell phenotypic transformation and neuro-inflammation after intracerebral hemorrhage in mice. Critical care medicine, 44(6), e390-e402.

Publication 2016

CM3050S Anti-PDGFR-β antibody anti-p-p38 MAPK anti-ICAM-1 anti-SMemb appropriate fluorescence conjugated secondary antibodies MagnaFire SP 2.1B

Actin Anesthesia Anti antibody Antibodies Brains Cold Fluorescence Fluorescence microscope Formalin Frozen Icam 1 Immunofluorescence Mice Olympus P38 mapk Pdgfr β Phosphate Saline Smooth muscle Sucrose

Corresponding Organization :

Other organizations : Loma Linda University, Soochow University, First Affiliated Hospital of Soochow University, Army Medical University, Southwest Hospital, Second Affiliated Hospital of Zhejiang University

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Variable analysis

independent variables

Time post-ICH (24 hours)

dependent variables

Expression of PDGFR-β
Expression of p-p38 MAPK
Expression of p-MK2
Expression of ICAM-1
Expression of SMemb

control variables

Mice were perfused under deep anesthesia
Brains were fixed in 10% formalin at 4°C for a minimum of 3 days
Samples were dehydrated with 30% sucrose in phosphate-buffered saline (PBS, pH 7.4)
Frozen coronal slices (10 μm thick) were sectioned in cryostat
Primary antibodies were incubated overnight at 4°C
Appropriate fluorescence conjugated secondary antibodies were used

positive controls

None specified

negative controls

None specified

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