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PubMed2004

[Detection of EWS-FLI1 fusion transcript in Ewing's sarcoma/peripheral primitive neuroectodermal tumors by one-step RT-PCR using paraffin-embedded tissues].

OBJECTIVE: To investigate the expression of EWS-FLI1/ERG fusion transcript resulting from t(11;12)(q24;12) in paraffin-embedded tissues and its diagnostic implication for Ewing's sarcoma/peripheral primitive neuroectodermal tumors (ES/pPNET). METHODS: One-step reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to detect a characteristic EWS-FLI1/ERG fusion transcript in 25 cases of ES/pPNET and 15 cases of other small round cell tumors (including 8 cases of rhabdomyosarcoma, 4 cases of synovial sarcoma, 2 cases of neuroblastoma and 1 case of lymphoma) using formalin-fixed and paraffin-embedded tissues. RESULTS: EWS-FLI1/ERG fusion transcript was detected in 20 of the 25 ES/pPNET cases (80%). The 15 non-ES/pPNET control cases were negative for EWS-FLI1/ERG fusion transcript. CONCLUSIONS: Detection of EWS-FLI1/ERG fusion transcript is a reliable index for molecular diagnosis of ES/pPNET. One-step RT-PCR is a practical method for such analysis in routine paraffin-embedded tumor tissues.

Adolescent↗

PubMed2003

Two approaches to double post-embedding immunogold labeling of freeze-substituted tissue embedded in low temperature Lowicryl HM20 resin.

Double labeling is used for localizing two antigens simultaneously in the same tissue. We have used two approaches to post-embedding immunogold labeling to investigate whether nerve terminals in the guinea-pig anteroventral cochlear nucleus (AVCN) that contain gamma-aminobutyric acid (GABA) or glycine are capable of retrieving the other amino acid as part of an investigation of colocalization of these putative neurotransmitters. For this, vibroslices of perfusion-fixed brain stem were freeze-substituted and embedded in the low temperature resin, Lowicryl HM20. Simultaneous labeling of ultrathin sections was then performed with a mixture of a rabbit primary antibody to GABA and a guinea-pig primary antibody to the glycine transporter, GLYT2, followed by labeling with a mixture of secondary antibodies (goat anti-rabbit IgG-30 nm gold, goat anti-guinea pig IgG-15 nm gold). This approach indicated that GLYT2 occurs in the plasma membrane of some terminals that contain GABA. The other approach involved sequential labeling of ultrathin sections with a rabbit primary antibody to the GABA transporter, GAT1, followed by an anti-rabbit secondary antibody conjugated to 15-nm gold particles. Sections were then treated with paraformaldehyde vapor to denature any free anti-IgG binding sites on the first antibody, and labeled with a primary antibody to glycine also raised in rabbit followed by an anti-rabbit secondary antibody conjugated to 30-nm gold particles. This approach indicated that GAT1 occurs in the plasma membrane of some terminals that contain glycine. Thus, these techniques can be used to localize heat-labile multiple antigens in the same tissue.

Acrylic Resins↗

PubMed2000

The paraffin-embedded tissue blot detects PrP(Sc) early in the incubation time in prion diseases.

With the appearance of bovine spongiform encephalopathy (BSE) and a new variant of Creutzfeldt-Jakob disease (nvCJD) that seems to be caused by BSE, there is an increased need for improvement of diagnostic techniques and recognition of all variants of prion diseases in humans and animals. Publications on the immunohistochemical identification of PrP(Sc) in the tonsils and appendix in the incubation period of nvCJD indicate that new and more sensitive techniques for the detection of PrP(Sc) in various tissues may be a valuable tool for early diagnosis in prion diseases. We developed a new and sensitive technique to detect PrP(Sc) in formalin-fixed and paraffin-embedded tissue, the paraffin-embedded tissue blot (PET blot), and reinvestigated archival brain material from CJD as well as BSE and scrapie. In addition, C57/Bl6 mice experimentally infected with the ME7 strain were investigated sequentially during the incubation time to compare this new technique with conventional methodologies. The PET blot detects PrP(Sc) in idiopathic (sporadic) and acquired prion diseases, even in cases with equivocal or negative immunohistochemistry, and is more sensitive than the conventional Western blot and histoblot techniques. The PET blot makes possible the detection of PrP(Sc) during the incubation period long before the onset of clinical disease and in prion disease variants with very low levels of PrP(Sc). In mice experimentally infected with the ME7 strain, the PET blot detects PrP(Sc) in the brain 30 days after intracerebral inoculation-145 days before the onset of clinical signs. Its anatomical resolution is superior to that of the histoblot technique. It may therefore be of particular interest in biopsy diagnosis. Thus it complements other tissue-based techniques for the diagnosis of prion diseases in humans and animals.

Animals↗

PubMed2005

MALDI-TOF MS and TaqMan assisted SNP genotyping of DNA isolated from formalin-fixed and paraffin-embedded tissues (FFPET).

Formalin-fixed paraffin-embedded tissues (FFPET) from archived clinical samples provide an invaluable source for large-scale molecular genetic studies. Pharmacogenetic investigations that require long-term clinical follow-up data of patients may particularly benefit from FFPET analysis. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and TaqMan-based (Thermus aquaticus polymerase) methodologies have become standard genotyping procedures. However, no data are available on the applicability of MALDI-TOF MS to the genotyping of low quality DNA, as it is usually obtained from FFPET, and data from TaqMan genotyping are limited. We isolated constitutional DNA from 274 FFPET samples (229 patients with breast cancer and 45 patients with benign breast diseases) and genotyped 15 polymorphic loci in 10 genes. Nine SNPs were genotyped by MALDI-TOF MS, and six were genotyped by the TaqMan methodology. We established rates for successful allele assignment for all FFPET, for FFPET prepared prior to 1990, and for FFPET prepared post-1990. Both methodologies showed high success rates ranging between 70.9 and 99.6% (mean: 91.8%) for MALDI-TOF MS and between 82.3 and 97.7% (mean: 91.0%) for TaqMan genotyping. No significant differences in genotyping performances for FFPET prepared prior to 1990 or post-1990 were observed. With the exception of one, all other genotype frequencies were in Hardy-Weinberg equilibrium. Furthermore, genotype frequencies matched those observed in a German breast cancer population and other Caucasian populations. Our study shows for the first time that MALDI-TOF MS and TaqMan genotyping procedures provide reliable data, and are therefore applicable in studies that require large scale FFPET genotyping.

Alleles↗

PubMed1990

Immunophenotyping of cutaneous lymphoid infiltrates in frozen and paraffin-embedded tissue sections: a comparative study.

A panel of antibodies reactive in routinely fixed, paraffin-embedded tissue sections was compared with a panel of antibodies reactive in frozen sections for the immunophenotyping of cutaneous lymphoproliferative disorders. Three T cell-associated markers (UCHL1, MT-1, MT-2, six B cell-associated markers (MB-1, MB-2, LN-1, LN-2, L-26, 4KB5), immunoglobulin heavy and light chains, anti-LCA antibody, two markers for Reed-Sternberg cells (Ber-H2, Leu-M1), one marker for macrophages (Mac-387) and anti-S-100 protein antibody were tested on normal skin, inflammatory skin diseases, and cutaneous lymphomas and pseudolymphomas. On the basis of the results in frozen sections, 12 inflammatory T cell diseases, 14 T cell lymphomas and pseudolymphomas, and 10 B cell lymphomas and pseudolymphomas were identified. In addition, two cases of specific skin infiltrates of Hodgkin's disease have been examined. Among T cell markers, the greatest sensitivity was exhibited by UCHL1, which stained all but one specimen of T cell infiltrate; it was negative in one specimen of mycosis fungoides that progressed into a T-immunoblastic lymphoma. The combined use of MB-2, LN-2, and 4KB5 identified all B cell proliferations. LN-1 marked germinal centers in all cases of follicular lymphoma and pseudolymphoma. Ber-H2 stained the Reed-Sternberg cells in both cases of Hodgkin's disease and the large cells in the histiocytic type of lymphomatoid papulosis. Mac-387 and anti-S-100 protein antibody recognized macrophages and T-zone histiocytes (Langerhans cells and interdigitating cells), respectively. A panel of antibodies reactive in routinely fixed, paraffin-embedded tissue sections is proposed that facilitates the identification of most B and T cell infiltrates in the skin.

Antibodies, Monoclonal↗

PubMed1987

Evaluation of a method for controlled tissue embedding for histologic evaluation of tumor margins.

Controlled tissue embedding is a method which facilitates the histologic search of margins of excised skin and subcutaneous tissue for evidence of tumor extensions. Used on frozen unfixed specimens, data obtained from a study of 141 sections made from 32 excised tumors show that controlled embedding allows for adequate assessment of surgical margins in less time and with less tissue cutting as compared to conventional methods.

Basal Cell Carcinoma↗

PubMed1995

E-cadherin expression in bladder cancer using formalin-fixed, paraffin-embedded tissues: correlation with histopathological grade, tumour stage and survival.

To determine the potential prognostic value of epithelial cadherin (E-cadherin), a Ca(2+)-dependent cell-cell adhesion molecule, we have analysed its immunoreactivity and cellular localisation in 67 transitional cell carcinomas (TCC) using an avidin-biotin immunoperoxidase technique on formalin-fixed, paraffin-embedded tissues. These results were correlated with histopathological grade, tumour stage, presence of metastases and survival. In addition, 10 cystitis and 11 normal bladder biopsies were evaluated as controls. E-cadherin was expressed in a normal membranous pattern in all normal and 7 of 10 cystitis biopsies. Loss of normal surface E-cadherin expression was found in 3 of 15 superficial tumours and in 48 of 52 invasive cancers. Abnormal immunoreactivity was strictly related to tumour differentiation and stage. Fifteen of 20 well-differentiated (grade I) tumours showed preserved membranous E-cadherin immunoreactivity, while 46 of 47 moderate and poorly differentiated tumours (grades II and III) demonstrated abnormal staining patterns. Loss of membranous E-cadherin immunoreactivity was also associated with advanced tumour stage. There was a significantly higher 5-year survival rate for patients with preserved membranous staining compared with patients with abnormal staining.

Cadherins↗

PubMed1999

Comprehensive TP53-denaturing gradient gel electrophoresis mutation detection assay also applicable to archival paraffin-embedded tissue.

A comprehensive mutation detection assay is described for the entire coding region and all splice site junctions of TP53. The assay is based on denaturing gradient gel electrophoresis, which follows either multiplex polymerase chain reaction (PCR) applied to DNA extracted from fresh or frozen tissue samples or nested PCR applied to DNA extracted from paraffin-embedded tissue samples. In both instances, the analysis can be performed under a single set of conditions. When testing the assay on DNA from cultured lung cancer cell lines and from paraffin-embedded Dukes C colorectal carcinomas, significant TP53 mutations were observed at high frequencies in 15 of 16 lung cancer cell lines (94%) and in 21 of 30 paraffin-embedded tissue samples of Dukes C colorectal carcinomas (70%). A substantial proportion of these significant mutations occurred outside the evolutionary conserved region of TP53 in 4 of 16 lung cancer cell lines (25%) and in 11 of 30 paraffin-embedded colorectal carcinomas (37%). This underscores the importance of a comprehensive TP53 mutation analysis in those instances that TP53 mutation is taken into account for diagnostic and prognostic purposes.

Colorectal Neoplasms↗

PubMed1987

Flow cytometric analysis of nuclear DNA from adrenocortical neoplasms. A retrospective study using paraffin-embedded tissue.

Nuclear DNA content of paraffin-embedded tissue from 48 adrenocortical neoplasms, 18 histologically normal control adrenal glands, and five hyperplastic adrenal glands was analyzed retrospectively using flow cytometry. Aneuploidy was compared with morphologic criteria as a predictor of recurrence. All 18 controls, five hyperplastic glands, and 39 neoplasms were diploid. Nine neoplasms were aneuploid. Compared with their diploid counterparts, aneuploid neoplasms were more likely to weigh more than 50 g (P less than 0.0001) and to have three or more histologic features of carcinoma (P less than 0.0001). Thirty-six neoplasms were followed clinically for at least 2 years (range 24 to 120 months, mean = 64.6 months) or until local recurrence, metastasis, or death. Five were clinically malignant. Neoplasms which recurred or metastasized were more apt to be aneuploid (P less than 0.005) than those showing no evidence of further disease during the follow-up period. They were also more likely to weigh more than 50 g (P less than 0.005) and to have three or more histologic features of carcinoma (P less than 0.0025). However, neither aneuploidy, large size, nor unfavorable histology result was a consistent feature in every malignant neoplasm. Flow cytometric DNA content analysis appears to be as effective a predictor of clinical outcome as size and histology and may be of particular value when the morphologic features are ambiguous.

Adenoma↗

PubMed1998

PCR diagnosis on formalin-fixed, paraffin-embedded tissues with acid-fast stain and culture negativity in chronic dialysis patients of cervico-mediastinal tuberculous lymphadenitis.

BACKGROUND: Bacteriologic studies often provide negative results in tuberculous infection, and do not favour early diagnosis. Polymerase chain reaction (PCR) is known to diagnose tuberculosis quickly. With this in mind, we used PCR to detect mycobacterial DNA on formalin-fixed, paraffin-embedded tissues with acid-fast stain and culture negativity in two dialysis patients with cervico-mediastinal lymphadenopathy. METHODS: Sections of neck lymph nodes were cut at two different levels. At each level, two semi-adjacent sections with a thickness of 5 microm each were cut using standard microtomes with disposable blades. The first section mounted on a glass slide was stained by Ziehl-Neelsen, and the second section was examined by PCR based on a 123 bp fragment of IS6110 that is specific for the Mycobacterium tuberculosis complex. RESULTS: The histology of lymph nodes disclosed inflammatory necrotizing granulomas, but acid-fast stain for M. tuberculosis was negative in the two patients. DNA of M. tuberculosis was detected in lymph node samples from each patient by PCR on the IS6110 element and by dot-blot hybridization. CONCLUSIONS: PCR assay is a potentially useful approach for early and rapid diagnosis of tuberculous lymphadenitis in chronic dialysis patients, since mycobacterial staining and culture often provide negative results.

Adult↗

PubMed2003

Detection of ALK gene rearrangements in formalin-fixed, paraffin-embedded tissue using a fluorescence in situ hybridization (FISH) probe: a search for optimum conditions of tissue archiving and preparation for FISH.

BACKGROUND: It is widely known that the efficiency of fluorescence in situ hybridization (FISH) probes applied to formalin-fixed, paraffin-embedded tissues is affected by the conditions under which the tissues are fixed and embedded. However, relatively few studies address exactly how tissue archiving conditions affect the performance of FISH probes. We report our experience based on use of an ALK FISH probe, during the validation of its diagnostic utility. METHODS: We applied the probe to 77 formalin-fixed, paraffin-embedded tissue blocks archived from 1991 through to 2000, and studied the interrelationship between the archival age (which ranged up to 10 years), type and condition of tissue, duration required for optimum hydrolysis, and obtainability of hybridization signals. RESULTS: We found that as archival age and tissue collagen content increased, not only did hydrolysis times have to be prolonged in order to yield interpretable hybridization signals, but also the likelihood of blocks becoming non-signaling increased. The most striking positive correlations were seen between the archival age of signaling lymphoid blocks and their requisite hydrolysis times. CONCLUSIONS: The difficulty in applying FISH on archival tissue increases with its archival age and collagen content, and may necessitate changes in laboratory protocol accordingly.

Anaplastic Lymphoma Kinase↗

PubMed1999

Detection of PAX3-FKHR and PAX7-FKHR fusion transcripts in rhabdomyosarcoma by reverse transcriptase-polymerase chain reaction using paraffin-embedded tissue.

BACKGROUND: Alveolar rhabdomyosarcoma (RMS) is associated with a characteristic chromosomal translocation t(2;13)(q35;q14). The genes involved in this translocation are paired box (PAX)3 on chromosome 2 and forkhead in RMS (FKHR) on chromosome 13. An occasional variant translocation t(1;13)(p36;q14) affecting PAX7 and FKHR on chromosomes 1 and 13, respectively, has also been described. Chromosomal translocations in RMS are detected using conventional cytogenetic analysis, fluorescence in situ hybridization (FISH) or reverse transcriptase-polymerase chain reaction (RT-PCR) on fresh or frozen tissue samples. We describe the results of RT-PCR analysis of PAX3-FKHR and PAX7-FKHR chimeric messages in formalin-fixed, paraffin-embedded tissue samples from 17 RMS cases. METHODS: RNA was extracted from formalin-fixed, paraffin-embedded RMS tissue. Oligonucleotide primers corresponding to the regions of PAX3, PAX7 and FKHR were used for the detection of PAX3-FKHR and PAX7-FKHR chimeric messages. A seminested PCR of the PCR products was used to increase the sensitivity of detection. The amplified fragments were purified and directly sequenced to confirm the specificity of the methods. RESULTS: The PAX3-FKHR chimeric message was detected in all three cases of alveolar RMS but not in any of the 12 embryonal and two pleomorphic RMS cases. The PAX7-FKHR fusion transcript was detected in one case of embryonal RMS. CONCLUSIONS: The results indicate that the RT-PCR assay is a reliable method for the detection of the PAX3-FKHR fusion transcript of alveolar RMS in formalin-fixed, paraffin-embedded tissue. This simple method enables pathologists to identify chromosomal rearrangements in RMS as a diagnostic aid in cases where fresh or frozen tissue is not available.

DNA-Binding Proteins↗

PubMed1985

Preparation of monolayer smears from paraffin-embedded tissue for image cytometry.

A method is described for the preparation of monolayer smears from paraffin-embedded tissue suitable for automated image analysis and DNA measurements. The proposed technique uses enzyme treatment and syringing for cell dispersal. Slide preparation is performed by centrifugal cytology. After Feulgen staining the quality of the monolayer smears is sufficiently high to enable visual morphologic evaluation. Automated DNA measurements using the Leyden television analysis system (LEYTAS) show coefficients of variation (CV) of 4.5% for the diploid cell population of the suspended tissue. This is approximately the same as the CV in fresh material from the same tumor. Formalin fixed trout red blood cells are used as reference cells. By applying image cytometry to paraffin-embedded tissue this method allows retrospective studies of, for instance, the significance of DNA content with regard to the behavior of a tumor.

Animals↗

PubMed1996

The use of microwave irradiation as a pretreatment to in situ hybridization for the detection of measles virus and chicken anaemia virus in formalin-fixed paraffin-embedded tissue.

Microwave irradiation was investigated as a pretreatment to in situ hybridization on formalin-fixed, paraffin-embedded tissue. Two probe/tissue systems were used: a single-stranded RNA probe for the detection of measles virus nucleocapsid genome in subacute sclerosing panencephalitis brain tissue, and a double stranded DNA probe for chicken anaemia virus in thymus of chicken infected with the virus. Microwaving, when used as sole pretreatment, was not as effective as the more traditional enzyme pretreatments for in situ hybridization. However, when used in combination with existing pretreatments, a significant increase was found in hybridization signal in both brain and thymus tissue. This was emphasized when combination enzyme/microwave pretreatments were used prior to detection of measles virus by in situ hybridization in a series of five archival subacute sclerosing panencephalitis cases. The use of microwave irradiation would be recommended as a means of supplementing in situ hybridization methods, especially when using long-term formalin fixed paraffin-embedded tissue.

Animals↗

PubMed1998

Immunohistochemical analysis of mycosis fungoides on paraffin-embedded tissue sections.

Mycosis fungoides (MF) is typically characterized by dermal and epidermal infiltration of T lymphocytes with a helper/inducer phenotype. Immunophenotypic analysis of such cases was traditionally performed by flow cytometry or immunohistochemistry on cryostat sections. With the advent of new monoclonal antibodies developed against T-cell antigens, including CD3, CD4, CD5, and CD8, it is now possible to immunophenotype T-cell subpopulations in paraffin-embedded tissues. To investigate the potential use of these antibodies for the evaluation of cutaneous lesions, 35 specimens (34 skin and 1 lymph node) from 29 patients with MF were retrospectively reviewed and immunophenotyped in paraffin sections with antibodies to CD3 (T-cell CD3), CD4 (NCL-CD4-1F6), CD5 (NCL-CD5-4C7), CD8 (CD8/144B), and CD20 (L26). Epidermal and dermal distribution of T and B cells were analyzed, and we assessed the ratios of CD4+ to CD8+ T cells. All of our 35 cases demonstrated a predominant CD3+ T-cell population. In 32 cases, the neoplastic cells expressed CD3, CD4, and CD5 consistent with a T-helper/inducer phenotype. In three cutaneous cases, the neoplastic CD4+ T cells showed minimal or absent expression of CD5, indicating an aberrant phenotype. In the majority of cases, minimal CD8+ T cells were present in the background, but in four cases, the CD4:CD8 ratios were 2:1 or less. Thirty-two cutaneous cases demonstrated epidermotropism exclusively by CD4+ T cells; one case showed both CD4+ and CD8+ T cells. In 17 cutaneous cases, scattered dermal CD20+ B cells were found individually or in small clusters within the background surrounding the neoplastic infiltrates. We concluded, therefore, that the immunophenotypic analysis of T-cell subpopulations using monoclonal antibodies of CD3, CD4, CD5, and CD8 was useful for histologic evaluation and confirmation of MF lesions in paraffin-embedded tissue. These antibodies might also provide an effective method of immunophenotyping other neoplastic and non-neoplastic T-cell populations in paraffin-embedded tissues.

Adult↗

PubMed2002

A new method to extract nuclei from paraffin-embedded tissue to study lymphomas using interphase fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) is difficult to accomplish using thin-sections of paraffin-embedded lymphoid tissue because of the high cellularity and truncated cells that interfere with accurate scoring of individual nuclei. We modified and tested a new technique to isolate individual nuclei from tissue cores of paraffin-embedded tissue processed with xylene, proteinase K, citric acid, and pepsin. The efficacy of this method to study paraffin-embedded tissue was investigated in six normal lymph nodes or tonsils and 32 malignant lymphomas including five mantle cell, five follicular, five Burkitt, five extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue, five anaplastic large-cell, and seven diffuse large B-cell. Fusion of CCND1 and IgH, BCL2 and IgH, c-myc and IgH, and MALT1 and API2 were detected using probes with a dual-fusion FISH strategy. Anomalies involving ALK and BCL6 were detected using break-apart FISH probes. FISH studies were successful for each of the 38 specimens. Chromosome anomalies were detected in each malignant specimen, but not in the normal lymphoid tissue. The correct chromosome anomaly was detected in 22 of 22 specimens with genetic abnormalities that were established by other genetic techniques. This FISH technique is useful to detect chromosome anomalies with high sensitivity and specificity in paraffin-embedded tissue and may provide important diagnostic and prognostic genetic information.

Burkitt Lymphoma↗

PubMed1997

Evaluation of a fluorescence-labelled oligonucleotide probe targeting 23S rRNA for in situ detection of Salmonella serovars in paraffin-embedded tissue sections and their rapid identification in bacterial smears.

A method for the detection of Salmonella based on fluorescence in situ hybridization (FISH) has been developed and applied for the direct detection of Salmonella in pure cultures and in formalin-fixed, paraffin-embedded tissue sections. On the basis of the 23S rRNA gene sequences representing all of the S. enterica subspecies and S. bongori, an 18-mer oligonucleotide probe was selected. The specificity of the probe was tested by in situ hybridization to bacterial cell smears of pure cultures. Forty-nine of 55 tested Salmonella serovars belonging to subspecies I, II, IIIb, IV, and VI hybridized with the probe. The probe did not hybridize to serovars from subspecies IIIa (S. arizonae) or to S. bongori. No cross-reaction to 64 other strains of the family Enterobacteriaceae or 18 other bacterial strains outside this family was observed. The probe was tested with sections of formalin-fixed, paraffin-embedded tissue from experimentally infected mice or from animals with a history of clinical salmonellosis. In these tissue sections the probe hybridized specifically to Salmonella serovars, allowing for the detection of single bacterial cells. The development of a fluorescence-labelled specific oligonucleotide probe makes the FISH technique a promising tool for the rapid identification of S. enterica in bacterial smears, as well as for the detection of S. enterica in histological tissue sections.

Animals↗

PubMed2006

Methacarn fixation--effects of tissue processing and storage conditions on detection of mRNAs and proteins in paraffin-embedded tissues.

In this study, we examined suitable conditions for tissue fixation with methacarn and ethanol dehydration and storage of paraffin-embedded tissues (PETs) on gene expression analysis. With fixation and dehydration of rat liver tissues for up to 16 h (overnight) and 1 week, respectively, at 4 degrees C, integrity of extracted total RNAs and polypeptides did not vary, the former integrity being constantly lower than that with unfixed frozen tissue, while protein yield was slightly reduced with increasing dehydration. Retained expression levels of mRNAs and proteins were mostly unaffected by the period of fixation but slightly fluctuated with the length of dehydration. When PETs were stored for up to 12 months, integrity of both total RNAs and polypeptides was retained at 4 degrees C but reduced at room temperature. Reduced expression levels of mRNAs and proteins were also noted by storage at room temperature after 12 and 3 months, respectively. However, neither tissue processing nor storage affected variability in either mRNA or protein levels among samples. Thus, the results suggest that, for gene expression analysis, tissues can be fixed with methacarn and dehydrated for at least 1 day and 1 week, respectively, and PETs can be stored for at least 12 months, but a temperature of 4 degrees C is preferable.

Acetic Acid↗