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PubMed1989

The prediction of recurrence in meningiomas. A flow cytometric study of paraffin-embedded archival material.

Despite the complete macroscopic excision of meningiomas, there is a significant rate of recurrence approaching 20% at 20 years. The prediction of recurrence by clinical and histopathological means is inadequate. Flow cytometric analysis of deoxyribonucleic acid (DNA) in meningiomas has shown a correlation between a high proliferative index based on tumor cell-cycle stage (%S + %G2/M) and clinically aggressive behavior. Accordingly, the DNA analysis of meningioma tissue may be of value in predicting recurrence of these tumors. To test this hypothesis, the DNA of paraffin-embedded archival tissue from known recurrent meningiomas was compared with an age- and sex-matched nonrecurrent group. Both groups had comparable follow-up periods. Forty patients with total macroscopic removal at the time of surgery were analyzed. The paraffin blocks of these tumors were retrieved and reclassified histologically according to the World Health Organization system. Sections were then taken for flow cytometric study. The DNA analysis showed that the proliferative index of the recurrent group was significantly higher than that of the nonrecurrent group (p less than 0.002), although the histological subtyping of the two groups was similar. These results support the suggestion that flow cytometry may be of value in the prediction of recurrence of histologically benign, macroscopically removed meningiomas.

Flow Cytometry↗
PubMed1986

Argyria: microanalytic-morphologic correlation using paraffin-embedded tissue.

Scanning electron microscopes, which have energy dispersive spectroscopy (x-ray microanalysis) capability, can provide accurate identification of the composition of inorganic deposits in tissue. We report a case of florid argyria in which the same deparaffinized 4-micron sections were sequentially examined by light microscopy, scanning electron microscopy, backscattered electron imaging, and x-ray microanalysis to confirm the clinical impression of argyria. We recommend the use of x-ray microanalysis of paraffin-embedded tissue as a simple, rapid and inexpensive means of precise identification of inorganic deposits of heavy elements in tissue sections.

Administration, Intranasal↗
PubMed1997

Detection of Mycobacterium bovis in formalin-fixed, paraffin-embedded tissues of cattle and elk by PCR amplification of an IS6110 sequence specific for Mycobacterium tuberculosis complex organisms.

A presumptive diagnosis of tuberculosis can be made if a tissue has characteristic histopathologic changes and acid-fast organisms. However, definitive diagnosis requires culture and species identification of the causative mycobacterium, a process that takes several weeks to complete. The purpose of work reported here was to determine if formalin-fixed, paraffin-embedded tissues could be tested by polymerase chain reaction (PCR) to provide a more rapid diagnosis of tuberculosis. Nondecalcified tissues from cases of tuberculosis in cattle and elk (Cervus elaphus) were examined. The primers used for PCR amplified a 123-bp fragment of IS6110, an insertion sequence that is specific for organisms in the Mycobacterium tuberculosis complex (M. tuberculosis, M. bovis, M. microti, M. africanum). The PCR test detected this sequence in tissues from 92 of 99 (93%) tuberculosis cases, including 3 of 4 elk. In 80 tissues, the positive results were obtained using material prepared by immersion of paraffin sections in water containing a detergent, followed by alternating boil/freeze cycles. The remaining positive results were obtained with DNA isolated from the crude tissue extracts by proteinase K digestion and phenol/chloroform purification. Accuracy of the IS6110 PCR test was demonstrated by negative test results on 31 tissues that had either nonmycobacterial granulomas or granulomatous lesions caused by other mycobacteria (M. paratuberculosis or M. avium). The findings of this study show that a PCR test usually can provide a rapid diagnosis of tuberculosis when it is applied to paraffin sections that have characteristic lesions and acid-fast organisms.

Animals↗
PubMed1991

Image analysis for quantitation of estrogen receptor in formalin-fixed paraffin-embedded sections of breast carcinoma.

Monoclonal antibody to human estrogen receptor (ER) provides a useful immunohistochemical tool for the evaluation of ER content in breast carcinoma, but visual interpretation is subjective. Computer-assisted image analysis has proved effective in immunohistochemical quantitation of ER in fresh tumor imprints and cryostat sections. We examined the usefulness of this technique in 5-microns-thick formalin-fixed paraffin-embedded tissue sections of 66 cases of primary breast carcinoma previously assayed by dextran-coated charcoal (DCC) analysis. Immunohistochemistry was automated and performed on a Code-on slide stainer (Instrumentation Laboratories, Lexington, MA) using Pronase predigestion, a monoclonal antibody (ER-ICA; Abbott, Chicago, IL), and a biotin-labeled secondary antibody. Detection was achieved with an avidin-alkaline phosphatase conjugate and nitroblue tetrazolium (NBT) bromochloroindoyl phosphate (BCIP) substrate. The immunohistochemical ER staining was analyzed visually and with the CAS/200 image analyzer (Elmhurst, IL). The visual semiquantitative histologic scores (HSCORE), the automated quantitative assays including the percentage of positive nuclear areas (PNA), and the quantitative immunocytochemical scores (QIC SCORE = PNA x % of positive stain/10) were compared with the corresponding DCC results. Linear correlations were demonstrated between all immunohistochemical assays and the logarithm of DCC, the strongest correlation seen with PNA (r = 0.91). Threshold points for positive HSCORE, QIC SCORE, and PNA assays were extrapolated using DCC as the reference. ER immunodetection by PNA as compared with visual examination alone was enhanced by 18% (up to 88%) in sensitivity and 34% (up to 94%) in specificity, and the DCC concordance rate increased by 26% (up to 91%). A comparative chart extrapolating DCC from PNA was thus established.(ABSTRACT TRUNCATED AT 250 WORDS)

Breast Neoplasms↗
PubMed2004

Quantitative gene expression profiling in formalin-fixed, paraffin-embedded tissues using universal bead arrays.

We recently developed a sensitive and flexible gene expression profiling system that is not dependent on an intact poly-A tail and showed that it could be used to analyze degraded RNA samples. We hypothesized that the DASL (cDNA-mediated annealing, selection, extension and ligation) assay might be suitable for the analysis of formalin-fixed, paraffin-embedded tissues, an important source of archival tissue material. We now show that, using the DASL assay system, highly reproducible tissue- and cancer-specific gene expression profiles can be obtained with as little as 50 ng of total RNA isolated from formalin-fixed tissues that had been stored from 1 to over 10 years. Further, tissue- and cancer-specific markers derived from previous genome-wide expression profiling studies of fresh-frozen samples were validated in the formalin-fixed samples. The DASL assay system should prove useful for high-throughput expression profiling of archived clinical samples.

Algorithms↗
PubMed2004

Prognostic significance of molecular upstaging of paraffin-embedded sentinel lymph nodes in melanoma patients.

PURPOSE: Detection of micrometastases in sentinel lymph nodes (SLNs) is important for accurate staging and prognosis in melanoma patients. However, a significant number of patients with histopathology-negative SLNs subsequently develop recurrent disease. We hypothesized that a quantitative realtime reverse transcriptase polymerase chain reaction (qRT) assay using multiple specific mRNA markers could detect occult metastasis in paraffin-embedded (PE) SLNs to upstage and predict disease outcome. PATIENTS AND METHODS: qRT was performed on retrospectively collected PE SLNs from 215 clinically node-negative patients who underwent lymphatic mapping and sentinel lymphadenectomy for melanoma and were followed up for at least 8 years. PE SLNs (n = 308) from these patients were sectioned and assessed by qRT for mRNA of four melanoma-associated genes: MART-1 (antigen recognized by T cells-1), MAGE-A3 (melanoma antigen gene-A3 family), GalNAc-T (beta1-->4-N-acetylgalactosaminyl-transferase), and Pax3 (paired-box homeotic gene transcription factor 3). RESULTS: Fifty-three (25%) patients had histopathology-positive SLNs by hemotoxylin and eosin and/or immunohistochemistry. Of the 162 patients with histopathology-negative SLNs, 48 (30%) had nodes that expressed at least one of the four qRT markers, and these 48 patients also had a significantly increased risk of disease recurrence by a Cox proportional hazards model analysis (P <.0001; risk ratio, 7.48; 95% CI, 3.70 to 15.15). The presence of > or = one marker in histopathology-negative SLNs was also a significant independent prognostic factor by multivariate analysis for overall survival (P =.0002; risk ratio, 11.42; 95% CI, 3.17 to 41.1). CONCLUSION: Molecular upstaging of PE histopathology-negative SLNs by multiple-marker qRT assay is a significant independent prognostic factor for long-term disease recurrence and overall survival of patients with early-stage melanoma.

Adult↗
PubMed1995

Immunohistochemical assay for epidermal growth factor receptor on paraffin-embedded sections: validation against ligand-binding assay and clinical relevance in breast cancer.

Epidermal growth factor receptor (EGFR) has been the subject of much research since it was first described as a prognostic factor in breast cancer. The assay methods used and results obtained vary widely between studies. In this study 88 primary breast cancers were assayed for EGFR using a novel immunohistochemical assay performed on paraffin-embedded sections. The monoclonal antibody used was raised against purified, denatured EGFR, reacts with an epitope on the external domain and does not interfere with ligand binding. Twenty-two per cent of the tumours were EGFR positive using this assay. The results obtained were significantly correlated with those obtained by ligand-binding assay (r = 0.621, P = 0.011). The concordance rate was 82% (P < 0.001). The majority of discordant results could be explained by the presence of benign breast tissue and other non-malignant elements which could be seen to express EGFR on the immunohistochemical assay and were excluded from the score for this, but would be incorporated into ligand-binding assay results. The well-established inverse relationship between EGFR (as measured by this assay) and oestrogen receptor (ER) was seen (chi 2 = 24.9, P < 0.0001). In addition, in this exploratory study on a limited tumour set, EGFR was a significant adverse prognostic factor (on univariate but not multivariate analysis) for both relapse-free survival (P = 0.02) and overall survival (P = 0.03) when measured by this immunohistochemical assay, but was not significant when measured by ligand-binding assay.

Adult↗
PubMed1979

Application of the aluminum-formaldehyde (ALFA) histofluorescence method for demonstration of peripheral stores of catecholamines and indolamines in freeze-dried paraffin-embedded tissue, cryostat sections and whole-mounts.

This paper describes new procedures for highly sensitive visualization of monoamine stores in peripheral tissues, taking advantage of the recently introduced aluminum-catalysed formaldehyde (ALFA) reaction. The tissues are exposed to an aluminum sulphate solution (with or without formaldehyde fixation) in a perfusion and/or immersion step, followed by formaldehyde vapour treatment. Procedures are described for freeze-dried, paraffin embedded tissue, cryostat sections and whole mount preparations. For all these tissue preparations the ALFA method gives a highly sensitive and precise demonstration of catecholamine-containing neurons and 5-HT-containing cells in a variety of peripheral tissues. For freeze-dried tissue and cryostat sections the ALFA method represents an improvement in comparison with other available methods. This is particularly noticeable for the very delicate adrenergic nerves in such organs as the thyroid, ovary, pancreas and the gastrointestinal tract.

Aluminum↗
PubMedDate not supplied

Detection of monokines in paraffin-embedded tissues of pigs using polyclonal antibodies.

Monokines are glycoproteins, synthesised by macrophages, which exert various effects on the organism. The most important monokines are interleukin (IL)-1alpha, IL-1beta, tumor necrosis factor (TNF)-alpha and IL-6. This paper reports on immunohistochemical techniques developed for the detection of IL-alpha, IL-1beta, IL-6 and TNF-alpha in fixed and paraffin-embedded pig tissues (spleen, lymph nodes, thymus, liver and kidney). Different fixatives (buffered formalin, acetic formalin, paraformadehyde-lysine-periodate and Bouin solution), and antigen unmasking techniques (permeabilisation with Tween 20, pronase enzymatic digestion and microwave-citrate buffer) were used. We describe different protocols for detection of monokines using polyclonal antibodies against the studied monokines. No signal was obtained with monoclonal antibodies against pig-TNF-alpha and human IL-1alpha. Bouin solution was shown to be the best fixative for immunohistochemical detection of IL-1alpha, TNF-alpha, and IL-6, using permeabilisation with Tween 20 as an unmasking antigen method. Acetic formalin was shown to be the best fixative for IL-1beta detection, not needing antigen retrieval techniques. Macrophages were identified as the main cytokine-producing cells, although other types of cells also stained positively to some cytokines. These techniques represent valuable tools for studies of the pathogenesis of viral and bacterial diseases, and of the immune system of the pigs.

Animals↗
PubMed1988

DNA ploidy patterns in cervical intraepithelial neoplasia grade III, with and without synchronous invasive squamous cell carcinoma. Measurements in nuclei isolated from paraffin-embedded tissue.

This study presents the results of cytophotometric (CPM) and flow cytometric (FCM) DNA ploidy measurements in cervical intraepithelial neoplasias grade III (CIN III) with and without synchronous invasive squamous cell carcinoma. Hysterectomy and biopsy material from 21 patients 35 years of age or younger and from 18 patients age 50 years or older was studied. The DNA analysis was performed in nuclei isolated from specific areas of paraffin-embedded tissue. There were significant differences in the distribution of DNA patterns between the two age groups. About 80% of CIN III lesions in women 50 years of age or older, with or without a coexisting invasive cancer were aneuploid. In the group of younger women a diploid DNA pattern was found in about 60% of CIN III with concomitant invasive cancer. In the absence of an invasive cancer, CIN III lesions were mostly polyploid. The DNA pattern of invasive cancers was generally identical with the adjacent CIN, thus suggesting that the two lesions were related. Although the prognostic value of DNA ploidy measurements in cervical intraepithelial lesions in women in these two age groups has to be further evaluated, these results are at considerable variance with previously published data on DNA values in CIN and invasive carcinoma. In four CIN III lesions without invasive cancer, in women of the group of 35 years of age or younger, human papilloma virus common antigen could be demonstrated by immunochemical procedure. In three of these cases a polyploid DNA pattern was present; the fourth case showed a bimodal aneuploid pattern.

Adult↗
PubMed2005

Gene expression profiles in paraffin-embedded core biopsy tissue predict response to chemotherapy in women with locally advanced breast cancer.

PURPOSE: We sought to identify gene expression markers that predict the likelihood of chemotherapy response. We also tested whether chemotherapy response is correlated with the 21-gene Recurrence Score assay that quantifies recurrence risk. METHODS: Patients with locally advanced breast cancer received neoadjuvant paclitaxel and doxorubicin. RNA was extracted from the pretreatment formalin-fixed paraffin-embedded core biopsies. The expression of 384 genes was quantified using reverse transcriptase polymerase chain reaction and correlated with pathologic complete response (pCR). The performance of genes predicting for pCR was tested in patients from an independent neoadjuvant study where gene expression was obtained using DNA microarrays. RESULTS: Of 89 assessable patients (mean age, 49.9 years; mean tumor size, 6.4 cm), 11 (12%) had a pCR. Eighty-six genes correlated with pCR (unadjusted P < .05); pCR was more likely with higher expression of proliferation-related genes and immune-related genes, and with lower expression of estrogen receptor (ER) -related genes. In 82 independent patients treated with neoadjuvant paclitaxel and doxorubicin, DNA microarray data were available for 79 of the 86 genes. In univariate analysis, 24 genes correlated with pCR with P < .05 (false discovery, four genes) and 32 genes showed correlation with P < .1 (false discovery, eight genes). The Recurrence Score was positively associated with the likelihood of pCR (P = .005), suggesting that the patients who are at greatest recurrence risk are more likely to have chemotherapy benefit. CONCLUSION: Quantitative expression of ER-related genes, proliferation genes, and immune-related genes are strong predictors of pCR in women with locally advanced breast cancer receiving neoadjuvant anthracyclines and paclitaxel.

Adult↗
PubMed1985

Generation of monoclonal antibodies recognizing neuronal elements in formalin-fixed paraffin-embedded human tissue.

We used formalin-fixed human spinal cord and dorsal root ganglia as immunogens to generate monoclonal antibodies (mAb) which immunohistochemically react with neurons in formalin-fixed human tissue sections. Three of the mAb recognized all neuronal populations studied, including those in spinal cord, dorsal root ganglia, cerebellum, and cerebrum. A fourth mAb recognized neurons within spinal cord, dorsal root ganglia and dentate nucleus of cerebellum but not those in cerebrum or cerebellar hemispheres. This mAb, unlike the other three, did not recognize murine neurons. These data indicate the feasibility of generating mAb suitable for analysis of human pathological material in its most readily available form, formalin-fixed paraffin-embedded tissue.

Antibodies, Monoclonal↗
PubMed1992

Estrogen and progesterone receptor detection in archival formalin-fixed, paraffin-embedded tissue from breast carcinoma: a comparison of immunohistochemistry with the dextran-coated charcoal assay.

The steroid receptor (estrogen (ER) and progesterone receptor (PR] status was studied in 94 cases of invasive breast carcinoma from three separate institutions. All cases had fresh tissue examined for ER and PR by the dextran-coated charcoal cytosolic assay (DCC), and each case was examined immunohistochemically for ER and PR from archival formalin-fixed, paraffin-embedded tissue. Immunohistochemical assays (IH) were reviewed blinded to the DCC results and scored in a semiquantitative fashion prior to comparison to the DCC results. Overall, there was agreement between DCC and IH in 89% of ER and in 87% of PR assays. Some 50% of the ER discorrelations were of the IH-positive DCC-negative type, while 27% of the PR discorrelations were of this type. In four cases, both ER and PR did not correlate between IH and DCC determinations, with two being IH (ER and PR) positive and DCC negative, and two of the opposite type. The results of the study show that steroid receptor assays performed on routinely processed formalin-fixed archival material are reliable and closely recapitulate the results of traditional biochemical assays. Results suggest that, in the cases where IH is positive while DCC is negative, the IH result may actually provide a more reliable receptor status of the tumor than does the DCC result. Semiquantitation of fixed tissue IH assays shows a trend toward quantitative correlation with DCC results, but this correlation is weak, and factors concerning fixation and processing are most likely to be responsible.(ABSTRACT TRUNCATED AT 250 WORDS)

Breast Neoplasms↗
PubMed1992

A rapid and simple procedure for the routine detection of ras point mutations in formalin-fixed, paraffin-embedded tissues.

The use of the polymerase chain reaction (PCR) to detect specific DNA sequences in small amounts of tissues or cells has become a widespread tool in the field of molecular biology. With the better understanding of the clinical significance of oncogene activations in human tumors, the application of PCR in a routine setting is rapidly gaining importance. We have developed a rapid and simple procedure for the detection of mutated ras oncogenes in routinely fixed, paraffin-embedded tissue samples. DNA is isolated from three 10 microns tissue sections by incubation with a nonionic detergent and proteinase K, and can be directly used for amplification by PCR. The amplified DNA fragments are then dot-blotted onto nylon membranes and are hybridized to radioactively labeled oligodeoxynucleotides, specific for each of the mutated ras sequences. After a selective washing procedure, only fully matched oligodeoxynucleotides remain bound to the membrane, thus revealing the nature of the sequences that were present in the starting material. With this method, the detection of point mutations in ras genes can be performed in a routine setting, and the results of the analyses can be available in as few as 3-4 days.

Base Sequence↗
PubMed1996

Estrogen receptor immunocytochemistry in paraffin embedded tissues with ER1D5 predicts breast cancer endocrine response more accurately than H222Sp gamma in frozen sections or cytosol-based ligand-binding assays.

BACKGROUND: Historically, estrogen receptor (ER) determinations have been made by the ligand-binding assay of tumor homogenates, primarily by the dextran-coated charcoal method (DCC). Immunocytochemical assays (ICA) for ER are more recent and have been executed mostly on frozen sections with the monoclonal antibody H222Sp gamma (H222). Lately, new monoclonal antibodies derived by recombinant ER technology have been developed that work well on paraffin embedded, formalin fixed tissue sections. However, there is little information as to whether such assays prognosticate endocrine response. METHODS: Using antigen retrieval, the immunoglobulin G1 monoclonal antibody ER1D5, and the streptavidin-biotin detection system, 74 patients with breast cancer in whom endocrine response was known were assayed and the results compared with ER by DCC and ER by ICA in frozen section with H222. RESULTS: ER1D5 in paraffin provided the highest correlation with endocrine response (Kendall's tau [r] = 0.57; P<0.001) whereas ER by DCC failed to correlate (r= -0.002; P<0.99). ER1D5 in paraffin correlated weakly though significantly with DCC (Kappa Statistic [K] = 0.204; P<0.02). H222 in frozen sections also correlated moderately with endocrine response (r = 0.34; P<0.001). CONCLUSIONS: ER can be detected in routine tissue sections processed with antigen retrieval and ER1D5, and can be relied upon to provide accurate prognostic information regarding response to endocrine therapies in breast cancer patients.

Adult↗
PubMed2003

Optimization of biotinyl-tyramide-based in situ hybridization for sensitive background-free applications on formalin-fixed, paraffin-embedded tissue specimens.

BACKGROUND: Over the past five years in situ hybridization techniques employing tyramide amplification reagents have been developed and promise the potential detection of low/single-copy nucleic acid sequences. However the increased sensitivity that tyramide amplification brings about may also lead to problems of background staining that confound data interpretation. METHODS: In this study those factors enabling background-free biotinyl-tyramide based in situ hybridization assay of formalin-fixed paraffin-embedded tissues have been examined. SiHa, HeLa and CaSki cell lines known to contain HPV integrated into the cell genome, and archival cervical pre-invasive lesions and carcinomas have been successfully assessed using biotinylated HPV and centromeric probes. RESULTS: The single most important factor both for sensitivity and clean background was a tissue unmasking regimen that included treatment with 10 mM sodium citrate pH 6.0 at 95 degrees C followed by digestion with pepsin/0.2 M HCl. Concentrations both of probe and primary streptavidin-peroxidase conjugate and pH of hybridization mix and stringency washes were also critical for sensitivity. Certain probes were more associated with background staining than others. This problem was not related to probe purity or size. In these instances composition of hybridization mix solution was especially critical to avoid background. 3-amino-9-ethylcarbazole was preferred over 3,3'-diaminobenzidene as a chromogen because background was cleaner and the 1-2 copies of HPV16 integrated in SiHa cells were readily demonstrable. HPV detection on metaphase spreads prepared from SiHa cells was only successful when a fluorescent detection method was combined with tyramide reagent. 'Punctate' and 'diffuse' signal patterns were identified amongst tissues consistent with the former representing integration and 'diffuse' representing episomal HPV. Only punctate signals were detected amongst the cell lines and were common amongst high-grade pre-invasive lesions and carcinomas. However it remains to be determined why single/low-copy episomal HPV in basal/parabasal cells of low-grade lesions is not also detectable using tyramide-based techniques and whether every punctate signal represents integration. CONCLUSIONS: A tyramide-based in situ hybridization methodology has been established that enables sensitive, background-free assay of clinical specimens. As punctate signals characterize HPV in high-grade cervical lesions the method may have potential for clinical applications.

Journal Article↗
PubMed1988

Recognition of malignant melanoma by monoclonal antibody HMB-45. An immunohistochemical study of 200 paraffin-embedded cutaneous tumors.

Antibodies to S-100 protein have been used widely as markers of malignant melanoma, despite abundant evidence that they are non-specific for this neoplasm. Hence, alternatives to these reagents are desirable in diagnostic dermatopathology. We evaluated the characteristics of a new monoclonal antibody (HMB-45) which does have putative specificity for melanoma, and compared it with a polyclonal anti-S-100 reagent in immunohistochemical staining of 67 melanomas of the skin and 133 non-melanomatous cutaneous neoplasms. All specimens were formalin-fixed and paraffin-embedded, and were studied with the avidin-biotin-peroxidase complex technique. HMB-45 labelled 62 of 67 melanomas, while anti-S-100 recognized all tumors of this type. On the other hand, S-100 also was expressed by 15 of the non-melanocytic neoplasms, all 133 of which were HMB-45-negative. The only cases of melanoma that were missed by the latter reagent were of the spindle-cell type. Hence, HMB-45 was 100% specific and 93% sensitive, relative to a diagnosis of malignant melanoma in paraffin sections. Epithelioid and small-cell neoplasms are reliably recognized by this antibody, but it would appear that spindle-cell melanomas must be detected by other immunohistochemical means.

Antibodies, Monoclonal↗
PubMed1991

In situ hybridization for the detection of feline interleukin 1 alpha mRNA on the paraffin-embedded section using biotin-labeled probes.

In situ hybridization (ISH) technique with a biotin-labeled probe was established for detecting feline interleukin 1 (IL-1) alpha mRNA in necropsied specimens. Homology between human IL-1 alpha cDNA used as a probe and feline IL-1 alpha mRNA was confirmed by means of dot blot hybridization using the biotin-labeled probe. Hence, we tried by this biotinylated probe to detect mRNA of IL-1 alpha in paraffin-embedded sections. The following results were obtained for the routine procedures: 1) coating slides with poly-L-lysine and/or heating at 60 degrees C at least for 6 hours gave an excellent result for the adhesion of the tissue sections, 2) 10 micrograms/ml solution of proteinase K treatment for 30 minutes or 50 to 100 micrograms/ml solution of proteinase K treatment for 10 to 30 minutes at 37 degrees C gave the good results in the detection of ISH signal, 3) suitable denaturation time of probes at 70 to 90 degrees C was 5 to 15 minutes, and 4) effective hybridization was obtained by incubation for 24 hours at 4 degrees C, for 18 to 24 hours at 25 degrees C or for 5 to 24 hours at 37 degrees C.

Animals↗