Immunohistochemical Analysis of Corneal Tissue

Corneas were paraffin-embedded and cut in 5μm serial sections [28 (link)], then the paraffin was removed by a xylene substitute (Hemo-De; Thermo-Fisher Scientific, Darmstadt, Germany). The immunohistochemistry procedure was performed as previously described [24 (link)] by BenchMark Automated IHC/ISH slide staining system (BenchMarkVentana, Tucson, AZ, USA). Sections were incubated with specific anti-ki67 (Rabbit polyclonal anti-Ki67 antibody; concentration 5 μg/mL; ab155807 Abcam) and anti-VEGF (Mouse monoclonal anti-VEGF antibody; 1:100; sc-57496, Santa Cruz) antibodies. After washes with PBE, the section was incubated with biotin-conjugated secondary antibodies and avidin-biotin-peroxidase complex (DBA, Milan, Italy). Specific antigens in each section were located with 3,3′diaminoenzidine (DAB) reaction, then slides were counterstained with hematoxylin. An expert pathologist analyzed the immunostaining (intraobserver variability 5%). Ki67 and VEGF antigenic expression was measured and calculated by Leica IM500 and statistics program Leica QWIN (Leica, Wetzlar, Germany). Four distinct preparations for each corneal sample were done and 18 fields of view were analyzed in each preparation for a total area of 1.35631e ± 0.003 μm².

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Iannotta M., Belardo C., Trotta M.C., Iannotti F.A., Vitale R.M., Maisto R., Boccella S., Infantino R., Ricciardi F., Mirto B.F., Ferraraccio F., Panarese I., Amodeo P., Tunisi L., Cristino L., D’Amico M., di Marzo V., Luongo L., Maione S, & Guida F. (2021). N-palmitoyl-D-glucosamine, A Natural Monosaccharide-Based Glycolipid, Inhibits TLR4 and Prevents LPS-Induced Inflammation and Neuropathic Pain in Mice. International Journal of Molecular Sciences, 22(3), 1491.

Publication 2021

Hemo-De sc-57496 Leica IM500 Leica QWIN

Anti antibody Antibodies Antigenic Avidin Biotin Corneal Hematoxylin Hemo de Immunohistochemistry Mouse Paraffin Pathologist Peroxidase Rabbit Vegf Xylene

Corresponding Organization : Istituto Neurologico Mediterraneo

Other organizations : National Research Council, Université Laval

Top 5 similar protocols

Variable analysis

independent variables

Incubation of sections with anti-ki67 antibody
Incubation of sections with anti-VEGF antibody

dependent variables

Ki67 antigenic expression
VEGF antigenic expression

control variables

Paraffin embedding of corneas
Cutting of sections in 5μm serial sections
Removal of paraffin using xylene substitute
Immunohistochemistry procedure performed as previously described
Incubation with biotin-conjugated secondary antibodies and avidin-biotin-peroxidase complex
Visualization of specific antigens with 3,3′diaminoenzidine (DAB) reaction
Counterstaining of slides with hematoxylin
Analysis of immunostaining by an expert pathologist with intraobserver variability of 5%
Measurement and calculation of Ki67 and VEGF antigenic expression using Leica IM500 and Leica QWIN software
Four distinct preparations for each corneal sample and 18 fields of view analyzed in each preparation for a total area of 1.35631e ± 0.003 μm^2

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