Softening paraffin-embedded plant tissues.
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A method of sample preparation for electron microscope is introduced. After paraffin-embedded tissues, fresh and fixed on time, were deparaffined, the tissue were fixed in 4% glutaraldehyde for 24 hours, in 1% osmic acid for 1-2 hours, and dehydrated with acetone and embedded in Epon812; then they were cut into ultrathin sections. By this method, better ultrastructure of paraffin-embedded tissues was obtained, and the method could satisfy the demand for observation under electron microscope. In addition, some important proceedings were also discussed in the paper.
A fluorescent in situ hybridization (FISH) study was carried out on paraffin sections of human embryonic and fetal tissues with two DNA probes, DXZ1 and DYZ1 (Oncor), for X and Y chromosome-specific DNA sequences, respectively. The specificity of the DNA probes was confirmed on metaphase and interphase lymphocytes of healthy normal adult males. The paraffin blocks of the human embryonic and fetal tissues examined in the present study had been stored at room temperature for up to 5 years after fixation in 4% paraformaldehyde. All the seven embryos and five fetuses examined were successfully sexed by FISH. The cells from three embryos and four fetuses were positive for a hybridization signal with each of the DXZ1 and DYZ1 probes and they were classified as male. The cells from the remaining four embryos and one fetus were positive for two identical hybridization signals with the DXZ1 probe in a nucleus instead of the absence of the signal hybridized with DYZ1, indicating that their cells have two X chromosomes but no Y chromosomes. The FISH results for the five fetuses examined were consistent with their genital sex and/or gonadal histology. The FISH results were highly specific and no false positive or false negative results were obtained. Thus, the FISH technique has been shown to visualize specific DNAs in situ on paraffin sections and to be useful to determine the sex of fixed embryos and fetuses retrospectively.
An in situ DNA hybridization technique was used to identify various types of Human papillomavirus (HPV) in paraffin sections of serial punch biopsies taken from 64 patients having colposcopy because of abnormal smears. There women were in fact 64 consecutive patients out of 505 attending our clinic (at 6-month intervals) since 1981 for HPV infections. HPV 6 DNA sequences were found in 20%, HPV 11 in 17%, HPV 16 in 8% and HPV 18 in 5% of the 64 biopsies analysed with this method so far. More than 60% of HPV 6-positive lesions belong to HPV-NCIN (HPV lesion without concomitant CIN) or HPV-CIN I categories, as contrasted with HPV 16-positive lesions, 80% of which belong to HPV-CIN II and III categories. None of the HPV 16- or HPV 18-infected lesions regressed, as contrasted with 23% and 45% in those infected with HPV 6 and HPV 11, respectively (P less than 0.01). The rate of progression (38.4% and 45.5%, respectively) was markedly lower in HPV 6- and HPV 11 lesions as compared with that (80%) of HPV 16 lesions. The present results while supporting the concept on HPV 16 and HPV 18 as the high risk HPV types in cervical carcinogenesis also emphasize the applicability of the in situ DNA hybridization as a powerful tool in analysis of the specific HPV DNA sequences in routinely progressed biopsies of these lesions.
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Specific immunostaining of p21 ras protein by the well-characterized pan-ras antibody Y13-259 is achieved in paraffin sections of human and animal tissues fixed in periodate-lysine-paraformaldehyde-dichromate (PLPD). Intensity of staining is as good as in cryostat sections, with superior histological detail. Localization to plasma membrane is demonstrated in rodent cells genetically manipulated to express abundant p21 ras (the FHO5T1 cell line), both in preparations suspended in agar after culture in vitro and in those growing as tumour in vivo. Strong positive staining is observed in neoplasms of human breast and colon, tissues in which there is independent evidence of elevated ras gene expression. The superior morphology afforded by this technique allows clear characterization of p21 ras expression in small premalignant lesions for which other methods of detection of oncogene expression are not appropriate.
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