Patients
This study is a case-control-study involving 58 skin specimens divided into two groups. The first group involved 29 specimens from patients suffering from IAV and the second one 29 from non lesional skin of same patient, used as a control. Acne severity was graded as mild, moderate and severe according to the American Academy of Dermatology Consensus statement on acne classification [12]. Four to five mm skin biopsies were taken from the papular lesion after obtaining patients' consent. Patients were ≥ 15 years old untreated or their treatment was stopped for at least two months before the biopsies, and without systemic or other inflammatory skin diseases. Complete medical history, family history of acne and previous treatment received were assessed.
Biopsies and pathological examination
Biopsies were taken under local anaesthesia and were immediately embedded in Tissue Tek OCT compound (Miles Inc., Elkhart, Indiana, USA). Five μm thick cryostat sections were cut from tissue blocks and placed on super frosted slides. Sections were air dried for 3 hours. Slides were wrapped back to back in aluminum foil and stored frozen at -70°C until the time of staining. The rest of the biopsy was fixed in 10% neutral buffered formalin and processed to paraffin blocks. Haematoxylin and eosin (H&E) stained sections were assessed to evaluate the histopathological changes. The extent of inflammatory cells and dermal blood vessels were semi-quantitatively assessed as mild, moderate and severe, compared to the control group.
Immunohistochemistry
IL-8 utilized in the study is a mouse monoclonal IgG2b antibody raised against a recombinant protein corresponding to amino acids 40–99 mapping at the carboxy terminus of IL-8 of human origin (Santa Cruz, sc 8427). All incubations were done at room temperature.
Prior to staining, sections were brought to room temperature. Tissue sections were fixed in acetone for 10 minutes, air dried and submerged in phosphate buffered saline (PBS) bath for five minutes, before the start of staining. Excess buffer was tapped off followed by the incubation for 60 minutes with the primary antibody diluted at 1:100. Slides were washed twice with PBS for 5 minutes. The Dakocytomation, LSAB 2 was used as detection kit. Biotinylated secondary antibody was applied to the tissue sections for 15 minutes. After washing with PBS, streptavidin was applied for 15 minutes. Slides were washed with PBS then incubated with diaminobenzidine (DAB) chromogen for 10 minutes. Slides were rinsed with distilled water, counterstained with Harris hematoxylin (Hx), dehydrated and finally mounted. Negative controls were slides stained by omission of the primary antibody.
Staining interpretation
Cytoplasmic staining was considered positive. The intensity of staining was evaluated as 0) negative or absence of positive cells, 1) faint or mild, 2) moderate and 3) strong staining.
Statistical analysis
Categorical data were compared using Chi-square test and statistical significance was considered at p value
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