Immunofluorescence analysis of actinic keratosis

Actinic keratosis lesions treated with either metronomic or conventional PDT were harvested at 1-, 3- or 5-days post PDT. Formalin-fixed, paraffin embedded sections were evaluated by Hematoxylin/Eosin (H&E) staining. For immunofluorescent staining of protein markers, skin was embedded in Histochoice (VWR) rather than formalin. For immunofluorescence analyses of Caspase-3 expression and cleavage, the primary antibody specific to cleaved Caspase 3 was purchased from BioVision Inc. and antibodies for macrophages (F4/80) and neutrophils (Ly6G) were from BioRad and Affymetrix, respectively. The secondary antibodies, Cy3-conjugated donkey anti-rabbit IgG and Cy3-conjugated donkey anti-rat IgG were from Jackson ImmunoResearch. Relative expression of marker proteins in sections stained by immunofluorescence was analyzed using fluorescence microscopy at the Lerner Research Institute Image Core [29 (link), 30 (link)].

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Anand S., Yasinchak A., Govande M., Shakya S, & Maytin E.V. (2019). Painless versus conventional photodynamic therapy for treatment of actinic keratosis: Comparison of cell death and immune response in a murine model. Proceedings of SPIE--the International Society for Optical Engineering, 10860, 108600K.

Publication 2019

Histochoice F4/80) Cy3-conjugated donkey anti-rabbit IgG Cy3-conjugated donkey anti-rat IgG

Actinic keratosis Anti igg Antibodies Antibody Caspase 3 Cleavage Donkey Eosin Fluorescence microscopy Formalin Immunofluorescence Macrophages Neutrophils Paraffin Proteins Rabbit Skin

Corresponding Organization :

Other organizations : Cleveland Clinic, Cerner (United States), Dallas Plastic Surgery Institute

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Variable analysis

independent variables

Metronomic PDT treatment
Conventional PDT treatment

dependent variables

Expression of Caspase-3
Presence of macrophages (F4/80)
Presence of neutrophils (Ly6G)

control variables

Time points (1-, 3- or 5-days post PDT)
Formalin-fixed, paraffin embedded sections
Histochoice (VWR) embedding for immunofluorescence

controls

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

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