Immunofluorescence Staining of Wound Macrophages

Cytospin smears from wound mϕ suspension were fixed in cell fixation buffer (BD Cytofix, BD Biosciences, CA) for 10 min. Following fixation, wound mϕ were washed, blocked in 10% NGS for 30 min and were incubated in primary antibodies for Arginase-1 (1:100; BD Biosciences) and iNOS (1:100; Abcam,). Fluorescence tagged secondary antibody detection was performed with Alexa Fluor 568 secondary antibody (1:200, Life Technologies) as described previously^{25 (link)}.

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Das A., Datta S., Roche E., Chaffee S., Jose E., Shi L., Grover K., Khanna S., Sen C.K, & Roy S. (2018). Novel mechanisms of Collagenase Santyl Ointment (CSO) in wound macrophage polarization and resolution of wound inflammation. Scientific Reports, 8, 1696.

Publication 2018

BD Cytofix Arginase-1 Alexa Fluor 568 secondary antibody

Alexa 568 Antibodies Antibody Arginase 1 Buffer Cell Fluorescence Inos Wound

Corresponding Organization :

Other organizations : The Ohio State University Wexner Medical Center, Smith & Nephew (United States)

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Variable analysis

independent variables

Cytospin smears from wound mϕ suspension

dependent variables

Arginase-1 expression
INOS expression

control variables

Cell fixation buffer (BD Cytofix, BD Biosciences, CA)
Blocking with 10% NGS
Primary antibodies for Arginase-1 (1:100; BD Biosciences) and iNOS (1:100; Abcam)
Fluorescence tagged secondary antibody detection with Alexa Fluor 568 secondary antibody (1:200, Life Technologies)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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