Antigen recovery and preservation using the microwave irradiation of biological samples for transmission electron microscopy analysis.

Most studies using microwave irradiation (MWI) for the preparation of tissue samples have reported an improvement in structural integrity. However, there have been few studies on the effect of microwave (MW) on antigen preservation during sample preparation prior to immunolocalization. This report documents our experience of specimen preparation using an automatic microwave apparatus to obtain antigen preservation and retrieval. We tested the effects of MW processing vs. conventional procedures on the morphology and antigenicity of two different tissues: the brain and mammary gland, whose chemical composition and anatomical organization are quite different. We chose to locate the transcription factor PPARß/δ using immunocytochemistry on brain tissue sections from hamsters. Antigen retrieval protocols involving MWI were used to restore immunoreactivity. We also studied the efficiency of the ultrastructural immunolocalization of both PPARγ and caveolin-1 following MWI vs. conventional treatment, on mammary gland tissue from mice at 10 days of lactation. Our findings showed that the treatment of tissue samples with MWI, in the context of a process lasting just a few hours from fixation to immunolocalization, enabled similar, or even better, results than conventional protocols. The quantification of immunolabeling for cav-1 indicated an increase in density of up to three-fold in tissues processed in the microwave oven. Furthermore, MW treatment permitted the localization of PPARß/δ in glutaraldehyde-fixed specimens, which was impossible in the absence of MWI. This study thus showed that techniques involving the use of microwaves could largely improve both ultrastructure and immunodetection.

Expression of peroxisome proliferator activator receptor ß/δ (PPARß/δ) in acne vulgaris.

Sebum production is the key factor in the pathophysiology of acne. Studies in sebocyte and human sebaceous gland biology indicate that agonists of peroxisome proliferator-activated receptors (PPARs) alter sebaceous lipid production. Our objective was to detect the expression of PPARß/δ in acne lesions and find its contribution to disease pathogenesis. Twenty five acne vulgaris patients (14 males, 11 females) were included. In addition, 12 healthy volunteers (6 males, 6 females) served as controls. Punch biopsies (3mm) were taken from lesional skin of all patients, non-lesional skin in 12 patients, and from the healthy controls. The biopsies were estimated quantitatively for the level of PPARß/δ mRNA using reverse transcriptase-polymerase chain (RT-PCR) technique. PPARß/δ mRNA levels were significantly higher in patients than controls (p=0.00) and in patients' lesional than non-lesional skin (p=0.00). No significant difference however, was found between inflammatory and non-inflammatory lesions. Age and disease duration had no influence on mean PPAR mRNA levels in lesional skin. PPARß/δ is over expressed-in inflammatory and non-inflammatory acne vulgaris and may well be considered as a candidate target in future acne therapy. However, elucidation of its functional role is recommended.

Quantitative expression patterns of peroxisome proliferator-activated receptor-beta/delta (PPARbeta/delta) protein in mice.

The expression patterns of PPARbeta/delta have been described, but the majority of these data are based on mRNA data. To date, there are no reports that have quantitatively examined the expression of PPARbeta/delta protein in mouse tissues. In the present study, a highly specific PPARbeta/delta antibody was developed, characterized, and used to examine tissue expression patterns of PPARbeta/delta. As compared to commercially available anti-PPARbeta/delta antibodies, one of six polyclonal anti-PPARbeta/delta antibodies developed was significantly more effective for immunoprecipitation of in vitro-translated PPARbeta/delta. This antibody was used for quantitative Western blot analysis using radioactive detection methods. Expression of PPARbeta/delta was highest in colon, small intestine, liver, and keratinocytes as compared to other tissues including heart, spleen, skeletal muscle, lung, brain, and thymus. Interestingly, PPARbeta/delta expression was localized in the nucleus and RXRalpha can be co-immunoprecipitated with nuclear PPARbeta/delta. Results from these studies demonstrate that PPARbeta/delta expression is highest in intestinal epithelium, liver, and keratinocytes, consistent with significant biological roles in these tissues.

An immunohistochemical perspective of PPAR beta and one of its putative targets PDK1 in normal ovaries, benign and malignant ovarian tumours.

Peroxisome proliferator-activated receptor beta (PPAR beta) is a member of the nuclear hormone receptor family and is a ligand-activated transcription factor with few known molecular targets including 3-phosphoinositide-dependent protein kinase 1(PDK1). In view of the association of PPAR beta and PDK1 with cancer, we have examined the expression of PPAR beta and PDK1 in normal ovaries and different histological grades of ovarian tumours. Normal ovaries, benign, borderline, grades 1, 2 and 3 ovarian tumours of serous, muciuous, endometrioid, clear cell and mixed subtypes were analysed by immunohistochemistry for PPAR beta and PDK1 expression. All normal ovarian tissues, benign, borderline and grade 1 tumours showed PPAR beta staining localised in the epithelium and stroma. Staining was predominantly nuclear, but some degree of cytoplasmic staining was also evident. Approximately 20% of grades 2 and 3 tumours lacked PPAR beta staining, whereas the rest displayed some degree of nuclear and cytoplasmic staining of the scattered epithelium and stroma. The extent of epithelial and stromal PPAR beta staining was significantly different among the normal and the histological grades of tumours (chi(2)=59.25, d.f.=25, P<0.001; chi(2)=64.48, d.f.=25, P<0.001). Significantly different staining of PPAR beta was observed in the epithelium and stroma of benign and borderline tumours compared with grades 1, 2 and 3 tumours (chi(2)=11.28, d.f.=4, P<0.05; chi(2)=16.15, d.f.=4, P<0.005). In contrast, PDK1 immunostaining was absent in 9 out of 10 normal ovaries. Weak staining for PDK1 was observed in one normal ovary and 40% of benign ovarian tumours. All borderline and malignant ovarian tumours showed positive cytoplasmic and membrane PDK1 staining. Staining of PDK1 was confined to the epithelium and the blood vessels, and no apparent staining of the stroma was evident. Significantly different PDK1 staining was observed between the benign/borderline and malignant ovarian tumours (chi(2)=22.45, d.f.=5, P<0.001). In some borderline and high-grade tumours, staining of the reactive stroma was also evident. Our results suggest that unlike the colon, the endometrial, head and neck carcinomas, overexpression of PPAR beta does not occur in ovarian tumours. However, overexpression of PDK1 was evident in borderline and low- to high-grade ovarian tumours and is consistent with its known role in tumorigenesis.