Immunohistochemical Analysis of Acetylated Proteins

Paraffin-embedded eye samples were treated as described by Rossi et al. [12 (link)]. Eye sections were incubated with primary anti-acetyl-p53 and anti-acetyl-FOXO1 antibodies (Santa Cruz, USA) and with secondary goat anti-rabbit IgG (DBA, Milan, Italy). A negative control was performed with the primary antibody omitted (data not shown). Seven distinct tissue sections for each group of animals were done and 20 microscopic fields were analyzed in each section at 200x magnification, by an expert pathologist (intraobserver variability 6%) blinded to the experimental protocol. Of each total area, a computer-aided planimetry (IM500, Leica Microsystem, Milano, Italy) was performed and the percentage of positive stained area per total area analyzed was calculated. A color threshold mask for immunostaining was defined and applied to all sections.

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Rossi S., Di Filippo C., Gesualdo C., Testa F., Trotta M.C., Maisto R., Ferraro B., Ferraraccio F., Accardo M., Simonelli F, & D'Amico M. (2015). Interplay between Intravitreal RvD1 and Local Endogenous Sirtuin-1 in the Protection from Endotoxin-Induced Uveitis in Rats. Mediators of Inflammation, 2015, 126408.

Publication 2015

IM500

Animals Anti antibodies Anti igg Antibody Goat Microscopic Paraffin Pathologist Rabbit Tissue

Corresponding Organization :

Other organizations : University of Campania "Luigi Vanvitelli"

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Variable analysis

independent variables

Treatment of paraffin-embedded eye samples as described by Rossi et al. [12]

dependent variables

Percentage of positive stained area per total area analyzed for acetyl-p53 and acetyl-FOXO1 immunostaining

control variables

Eye sections
Primary anti-acetyl-p53 and anti-acetyl-FOXO1 antibodies (Santa Cruz, USA)
Secondary goat anti-rabbit IgG (DBA, Milan, Italy)
Microscopic fields analyzed at 200x magnification
Computer-aided planimetry (IM500, Leica Microsystem, Milano, Italy) to measure percentage of positive stained area per total area analyzed
Color threshold mask for immunostaining defined and applied to all sections
Expert pathologist (intraobserver variability 6%) blinded to the experimental protocol

controls

Negative control: Primary antibody omitted (data not shown)

Annotations

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