Detection of Papillomavirus Proteins and DNA in Paraffin-Embedded Tissue Sections

Abstract

The key events during the papillomavirus life cycle can be mapped in infected tissue samples by antibody detection and in situ hybridization. The ease of immuno-detection varies for different proteins and is dependent on antigen availability. Epitope exposure is sometimes necessary, because the antigen may become masked after formalin fixation and paraffin embedding of the infected tissue. Visualization of both nucleic acid and protein targets can be done simultaneously by combining in situ hybridization and immuno-detection methods.

Improved detection reveals active β-papillomavirus infection in skin lesions from kidney transplant recipients

“…This is fully consistent with what has been seen with other PV types, where the onset of vegetative viral genome amplification coincides closely with the expression of cytoplasmic E4 and nuclear L1 in some very superficial cells during productive infection. [49][50][51] In one highly differentiated actinic keratosis, the number of L1-positive nuclei was very high and most of them were in the thick layers of parakeratosis.…”

“…49 For the other markers analyzed (E4, MCM7, L1), antigen unmasking was performed by heating the slides in a conventional decloaking chamber, where they were placed for 10 min at 121 1C in 10 mM citrate buffer at pH 6.0 (Vector Laboratories, Burlingame, CA, USA). The immunofluorescent protein-protein double detection (HPV5 E4-MCM7) was carried out by incubating the slides overnight at 4 1C with the primary antibody anti-MCM7 (1:200) diluted in 5% normal goat serum (NGS), followed by amplification with TSA (Tyramide Signal Amplification; Perkin-Elmer, Waltham MA, USA); then, slides were incubated with the primary antibody anti-HPV5 E4 (1:1000) for 3 h and subsequently with the appropriate fluorescent secondary antibody.…”

“…50 The cellular proliferation marker MCM7 was also strongly increased in the lesion compared with the adjacent normal epithelium being most apparent in the basal and suprabasal layers, indicating that cells were stimulated to enter the cell cycle. 49,51 To investigate whether similar images of productive infection could be visualized in organ transplant recipients skin lesions, tissue specimens from the available formalin-fixed and paraffin-embedded blocks were costained using IF analysis with anti-E4 Table 3 summarizes all the lesions developed by the IF-positive patients and reports the details of the genotyping results for each formalin-fixed and paraffin-embedded block available, distinguishing the different parts of the lesions (edges, core or WL). As expected from the spectrum of the polyclonal anti-E4 antibody used, all the IF-positive sections harbored genotypes belonging to species 1.…”

“…In addition, increased MCM7 expression that extended into the upper epithelial layers was a common feature of all the E4-positive areas, indicating that cells were driven into the cell cycle in areas of productive viral infections. 23,49,50 The observed stimulation of basal cell proliferation may contribute, in association with other transforming agents, such as UVB irradiation, to the transformation process without necessarily being maintained in the more advanced disease.…”

Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor

“…The rafts were lifted onto stainless steel grids and were fed by diffusion from below with raft medium (3:1 [v/v] DMEM, F-12; 10% FBS, 0.4 μg/ml hydrocortisone, 0.1 nM cholera toxin, and 5 μg/ml transferring). Raft cultures were allowed to stratify for 11-17 days and were fixed in formalin for 4 hours, paraffin embedded, sectioned, and stained with H&E or by immunofluorescence as described by Peh et al (44). Abs used were mouse mAb anti-keratin 14 (Ab-1; Thermo Fisher Scientific) and polyclonal goat antiserum against filaggrin (N-20; Santa Cruz Biotechnology Inc.) and rabbit antiserum against involucrin (H-120; Santa Cruz Biotechnology Inc.).…”