Immunostaining of cell preparations: A comparative evaluation of common fixatives and protocols

Immunostaining of cytologic preparations has been beset by problems of inconsistency, high background staining, and the requirement of different fixatives for different antigens. This study sought to identify a universal fixative and a simple fixation protocol suitable for a wide range of tissue antigens commonly employed for cytologic diagnosis. In an analysis of 23 fixation protocols involving acetone, acetone/methanol, acetone/formalin, glutaraldehyde, ethanol, methanol, and formal saline, fixation in 0.1% formal saline overnight at 27°C followed by 10 min fixation in 100% ethanol produced the most consistent and optimal preservation of immunoreactivity which could be further enhanced by pre-treatment with microwaves for epitope retrieval. Blocking of endogenous peroxidase was not necessary with this fixation protocol. Provided the smears were well air-dried (for at least 14 hr) prior to immersion in formal saline, there was no need to employ adhesive-coated glass slides. The smears could be kept at 27°C (room temperature) for at least 7 days and at −70°C for 5 wk without loss of immunoreactivity as air-dried smears or after fixation in formal saline. One hundred percent acetone and 100% ethanol produced good morphology and immunoreactivity but a high level of background staining, whereas acetone-based mixtures resulted in inconsistent immunostaining. Diagn Cytopathol 1996;15:167–174. © 1996 Wiley-Liss, Inc.     

The Journal of Histotechnology Volume 9, March 1986 – December 1986

Abstract

We analyzed the relationship between tumor proliferation and expression of PCNA, whose role as a proliferative marker remains controversial. Bivariate DNA/PCNA flow cytornetric analysis of both clonogenic cell lines and several human tumor and normal samples were compared to Ki-67 immunostaining. Because there are 2 populations of PCNA in cells, a replicon-bound and a free nucleoplasmic form, PCNA labeling was strongly affected by the cell fixation procedure used: poor with paraformaldehyde, strong and uniform with methanol, and variable with acetone1 methanol. Only the acetonelmethanol fixation demonstrated good correlation between S-phase specificity and cell proliferation marker. Cells blocked in specific cell cycle phases were distinguished by different staining levels; PCNA expression was detectable from early G1, increased in late G1, reached a maximum in S, and remained high in G2M. Because of its long half-life, residual levels of PCNA were still detectable in G0, whereas expression of the proliferative marker Ki-67, expressed later in the cell cycle than PCNA, was very weak or even undetectable in G0 cells and in early G1 cells. Staining levels of PCNA in tumor cells were always higher than in normal cells whatever their origin. Similarly, resting normal lymphocytes displayed lower PCNA levels than those observed in leukemia lymphoblasts. Fewer normal cells stained with Ki-67: PCNA labeling tended to give an overestimation of the growth fraction. Comparison between PCNA and Ki-67 labeling showed a linear correlation; but when compared in S-phase fraction, Ki-67 performed better than PCNA. PCNA may be used with caution and attention to fixation. It may be difficult to distinguish between proliferative and newly quiescecnetl ls, because of its residual prolonged expression. (The J Histotechnol 22:287, 1999)     

Rapid fixation and immunofluorescent staining of cultured cells using microwave irradiation

Abstract

Microwave irradiation during tissue fixation and immunostaining reduces the sample preparation time. Microwave irradiation also facilitates the penetration of fixatives and antibody solutions into the tissues, resulting in efficient fixation and reduction of non-specific antibody binding, respectively. Experimental procedures involving immunofluorescence microscopy are time-consuming as this method relies on antigenantibody reaction. Here, we utilized a technique involving exposure of cultured cells and tissues to intermittent microwave irradiation and immunostaining of fixed samples. Intermittent microwave irradiation during fixation reduces the required incubation time with blocking and antibody solutions, and results in good preservation of the immunoreactivity of fixed cells. Microwave irradiation also reduces the non-specific binding of fluorescein-labeled antibodies. These microwave-assisted rapid immunofluorescence techniques are useful for analysis of molecular compositions in cultured cell systems.     

Delay in fixation does not affect the immunoreactivity of proliferating cell nuclear antigen (PCNA).

The effect of delayed fixation on the immunoreactivity of proliferating cell nuclear antigen (PCNA) was investigated using eight breast carcinomas. Topologically shuffled samples of each tumour were immersed in fixative at times of 0.5, 1, 2, 4, 6, 18, and 24 h after surgical removal. In addition to a PCNA index (percentage of positive cells per 1200 tumour cells), a semi-quantitative PCNA grading system was used, based on estimates of more than or less than 50 per cent of positive tumour cells at each time interval. The PCNA index of six tumours increased by a mean of 10 per cent with a fixation delay of 24 h. The PCNA grade of all eight tumours showed no change with delayed fixation.     

Expression of tyrosine kinase receptors Tie-1 and Tie-2 in giant cell tumor of the tendon sheath: a possible role in synovial proliferation

We have recently demonstrated that Tie-1 and Tie-2 are expressed in synovial cells from rheumatoid arthritis (RA). To elucidate the possible involvement of Tie receptors in synovial proliferation, we analyzed their expression by immunostaining in five cases of giant cell tumor of tendon sheath (GCTTS), which represents a proliferating lesion of synovial cells. Strong immunoreactivity for both Tie-1 and Tie-2, regardless of the individual patient's profile, was observed in all cases of GCTTS. Six sets of double immunohistochemical stainings for Tie-1/Tie-2 and fibronectin, CD68, or CD34 were carried out to determine the phenotype of Tie-1 and Tie-2-positive tumor components. In these studies, both Tie-1 and Tie-2 immunoreactivity were widely observed in the fibronectin-positive fibroblastic and the CD68-positive histiocytic mononuclear cells, as well as in the osteoclast-like giant cells. In tumor vasculature, Tie receptors were expressed in the CD34-positive endothelial cells possessing proliferating cell nuclear antigen (PCNA) immunoreactivity. We also evaluated the correlation of Tie-1/Tie-2 expression and proliferating cells in GCTTS by using double staining of Tie-1/Tie-2 together with PCNA. Overexpression of PCNA immunoreactivity was frequently found in Tie receptors-positive cells with no obvious differences in the expression pattern of Tie-1 and Tie-2. These findings suggest the possible involvement of Tie receptors in the pathogenesis of GCTTS other than solely via their involvement in angiogenesis and subsequent vascularization. It was demonstrated that Tie-2 immunoreactivity was restricted to the fibroblastic, but not histiocytic, phenotype in RA synovium, suggesting different regulatory control of Tie-2 expression in GCTTS and RA synovium. Overexpression of Tie receptors in GCTTS may imply a biological role for these receptors in synovial proliferation.     

Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: an index of cell proliferation with evidence of deregulated expression in some neoplasms

Proliferating cell nuclear antigen (PCNA) is a 36 kD nuclear protein associated with the cell cycle. A monoclonal antibody, PC10, that recognizes a fixation and processing resistant epitope has been used to investigate its tissue distribution. Nuclear PCNA immunoreactivity is found in the proliferative compartment of normal tissues. PCNA immunoreactivity is induced in lectin stimulated peripheral blood mononuclear cells in parallel with bromodeoxyuridine incorporation and the number of cells with PCNA immunoreactivity is reduced by induction of differentiation in HL60 cells. In non-Hodgkin's lymphomas a linear relation between Ki67 and PCNA staining was demonstrated. These data suggest that in normal tissues and lymphoid neoplasms, PCNA immunolocalization can be used as an index of cell proliferation. However, in some forms of neoplasia, including breast and gastric cancer and in vitro cell lines, the simple relation between PCNA expression and cell proliferation is lost. In some breast and pancreatic tumours there is apparent deregulation of PCNA with increased expression in tissues adjacent to the tumours. The over-expression in some tumours and in adjacent morphologically normal tissue may represent autocrine or paracrine growth factor influence on PCNA gene expression.     

Proliferating cell nuclear antigen in breast carcinomas

Proliferating cell nuclear antigen (PCNA), was examined by immunohistochemistry in 509 breast carcinomas. The immunoreactivity was found to be independent of the length of fixation when the tissue sections were microwaved before incubation with the primary antibody. The PCNA immunoreactivity was assessed by two semi-quantitative methods, which were correlated but not exchangeable. The comedo type of intraductal carcinomas and invasive ductal carcinomas had a higher PCNA score than other types. Lymph node metastases had a significantly higher PCNA score than primary carcinomas. High PCNA immunoreactivity was correlated with the presence of lymph node metastases, absence of tubule formation, numerous mitoses, severe nuclear pleomorphism, high histological grade and absence of progesterone receptors (PgR). PCNA in lymph node positive tumours was correlated with tumour type, especially with ductal carcinomas, absence of tubule formation, high histological grade and absence of PgR, whereas PCNA in lymph node negative tumours was correlated with large tumour size, numerous mitoses, severe nuclear pleomorphism and high histological grade. Number of mitoses and nuclear pleomorphism were the two most important factors in predicting the PCNA score; the absence of PgR and nuclear pleomorphism were important in lymph node negative and positive tumours, respectively. In a univariate analysis high PCNA score was found to be correlated with shorter relapse-free period and poorer over-all survival.     

Selected Enzyme Histochemical Techniques Facilitated by the Microwave Oven

Abstract

Enzyme procedures may require 60 minutes or more reaction time at room temperature. The microwave oven was used to produce a substrate solution internal temperature of 38°C, which allowed the demonstration of enzyme activity of acid phosphatase, alkaline phosphatase, alpha naphthyl acetate esterase, naphthol AS-D chloracetate esterase, and acetyl cholinesterase within a greatly reduced period. The procedures can be done on frozen sections and cold processed glycol methacrylate embedded tissues.     

Double Fixation with Paraformaldehyde and Acetone for Optimal Immunocytochemical Demonstration of DNA Polymerase α

Abstract

Because the fixation procedure is a crucial step for immunocytochemical staining of DNA polymerase α, we sought an optimal fixation for demonstration of the enzyme using HeLa S3 cells. Double fixtion with paraformaldehyde and acetone produced intense stainability when slides were dipped in a 4% paraformaldehyde solution for 10 min, then in acetone for 5 rnin at 4°C. In contrast, fixation with ethanol or methanol resulted in considerable reduction of immunoreactivity. Single fixation with paraformaldehyde or acetone yielded intermediate stainability. Poor stainability also resulted in decrease of positive cells. (The J Histotechnol 12:285, 1989)     

A study of cell proliferation in formalin-fixed, wax-embedded bone marrow trephine biopsies using the monoclonal antibody PC10, reactive with proliferating cell nuclear antigen (PCNA).

We have investigated proliferation in bone marrow trephine biopsies from 32 patients with normal or abnormal haemopoiesis, using the monoclonal antibody PC10, which detects proliferating cell nuclear antigen (PCNA), together with immunohistochemical markers of haemopoietic cell lineage. PCNA immunostaining revealed the pattern of proliferation within individual haemopoietic lineages in normal marrow. Two unexpected observations were made: of erythroid cells, only pro-erythroblasts and occasional early normoblasts reacted, and positivity of megakaryocytes was unrelated to nuclear lobulation or CD61 expression. The pathological cases represented conditions in which haemopoiesis is increased (reactive hyperplasia, chronic granulocytic leukaemia, myeloproliferative and myelodysplastic syndromes, megaloblastic anaemia). Increases in the number, and disturbances of the spatial organization, of PCNA-expressing cells were present to a variable extent in all cases. Sheets of PCNA-positive megaloblastoid erythrocytes were frequently found in myelodysplastic and myeloproliferative tissue, associated with marked disturbances in the spatial organization of all haemopoietic lineages. Cases of megaloblastic anaemia due to vitamin B12/folate deficiency also demonstrated greatly increased erythroid PCNA expression, with positivity in some giant metamyelocytes. In addition to reflecting increased proliferation, elevated PCNA expression in some bone marrow pathologies may be due to altered kinetics of the protein induced by disturbances in growth factor production.     

Expression of bcl-2 oncoprotein in transitional cell carcinoma of the upper urinary tract

 We investigated the immunoreactivity for bcl-2 oncoprotein in 154 cases of transitional cell carcinoma of the upper urinary tract (TCC-UUT) and its relation with the immunoreactivity for p53 oncoprotein and proliferating cell nuclear antigen (PCNA) immunoreactivity. Immunohistochemically, bcl-2 oncoprotein was recognized as positive in 29.2% of the samples. The immunoreactivity for bcl-2 oncoprotein was significantly (P < 0.05) correlated only with stage, though there was a borderline correlation (P = 0.050) with PCNA immunoreactivity. Furthermore, in invasive TCC the immunoreactivity for bcl-2 oncoprotein was associated with PCNA immunoreactivity (P < 0.041). The 5-year disease-free and overall survival rates were 55.7% and 71.5%, respectively. A univariate analysis of survival revealed that stage, grade, pattern of growth, immunoreactivity for p53 oncoprotein, and PCNA immunoreactivity each had a significant effect on disease-free and overall survival rates, whereas the immunoreactivity for bcl-2 oncoprotein had no significant effect on either rate. In the final models of the multivariate analysis, stage was found to be the only prognostic factor for disease-free survival and for overall survival. Detection of immunoreactivity for bcl-2 oncoprotein appears to be of no real value in deciding the prognosis of TCC-UUT. Received: 3 July 1997 / Accepted: 29 October 1997     

Immunoreactivity of proliferating cell nuclear antigen in salivary gland tumours: An assessment of growth potential

Immunoreactivity of proliferating cell nuclear antigen (PCNA) was assessed to evaluate growth potential in surgically resected tissue specimens from 70 cases of benign and malignant salivary gland tumours. Three stage streptavidin-biotin immunoperoxidase immunostaining using monoclonal antibody to PCNA showed a heterogeneity of PCNA index and distribution. In normal salivary gland specimens, PCNA was demonstrated in the nuclei of few ductal and acinar cells. In pleomorphic adenoma a multiple nodular growth pattern was observed with positive immunoreactivity restricted to the nuclei of tubulo-ductal structures. Warthin's tumour had positive nuclei in the outer cuboidal cells of epithelial component and germinal centres of lymphoid tissue. Myoepithelioma and acinic cell carcinoma showed slightly differing values and a statistically significant difference in the value of the index was observed in tumour cell aggregates of the cribiform type of adenoid cystic carcinoma and the solid undifferentiated type and between low/intermediate and high-grade mucoepidermoid tumours. PCNA is a useful marker of tumour cell proliferation; the index correlates with the grade of malignancy in salivary gland tumours.     

Patterns of integrin common chain β1 and collagen IV immunoreactivity in hepatocellular carcinoma. Correlations with tumour growth rate,grade and size

Thirty cases of hepatocellular carcinoma (HCC) were investigated immunocytochemically for expression of β1 integrin molecule and of collagen IV. Immunoreactivity was related to the tumour proliferation index, as detected by PCNA immunostaining, and to tumour size and grade. Membrane β1 integrin immunoreactivity was deteccted in the neoplasticc cells of all cases, though two different staining patterns were clearly recognized. In 14 cases, β1 integrin immunoreactivity was confined to the cell-stroma interface, showing the same polarized pattern as the non-neoplastic cell counterpart. This staining pattern was associated significantly (P 0.0001) with low PCNA labelling (i.e. less than 20 per cent of neoplastic cells showing nuclear immunostaining. Conversely, 16 cases showed non-polarized pericellular β1 integrin immunostaining. This staining pattern was significantly associated (P < 0.0001)) with high PCNA labelling (more than 20 per cent of immunoreactive cells) and with tumour size greater than 4 cm in diameter (P < 0.0001). β1 Integrin, collagen IV, and PCNA immunoreactivities, however, did not correlate with the histological grade. The data emphasize that neoplastic progression of HCCs may be correlated with an aberrant expression of adhesion molecules and with a disruption of the collagen IV complement of basal membranes.     

Induction of proliferating cell nuclear antigen and Ki-67 expression by cytomegalovirus infection

With the goal of facilitating viral reproduction, cytomegalovirus (CMV) induces changes in the host cell replication machinery. Very little information is available, however, on the effects brought about by CMV on proliferating cell nuclear antigen (PCNA) and Ki-67 expression in infected cells. Fifty-five paraffin-embedded tissue samples (43 gastrointestinal, 10 skin, and 2 kidney biopsies) with both histological and immunohistochemical evidence of CMV infection were investigated for PCNA and Ki-67 expression by the avidin– biotin–peroxidase method. Of the 55 cases studied, 47 were positive for PCNA and 46 for Ki-67. PCNA and Ki-67 immunostaining was more striking in CMV-immunoreactive, inclusion-free cell nuclei, whereas cell nuclei exhibiting well-developed CMV inclusions either showed a weak peripheral signal for both proliferation markers, or were completely negative. Enhanced PCNA and Ki-67 expression appears to be among the changes induced by CMV infection in host cells. Moreover, this induction seems to reach its peak during the earlier phases of CMV infection and abate as the infection proceeds to its inclusion-forming phases, when a sufficiently high viral load would have been attained. © 1998 John Wiley & Sons, Ltd.     

Assembly of a temperature-sensitive mutant of Rauscher murine leukemia virus at the cell surface induced by low temperature and by ligands.

NIH/3T3 mouse fibroblasts, infected with a Rauscher murine leukemia virus temperature-sensitive mutant (ts25) defective in assembly of budding particles at 39°, produce virus at the cell surface when the temperature is shifted rapidly to 0°. Virus buds are not assembled within the first 10 min at 0° and gradually increase in number and degree of development over a 2-hr period. However, release of infectious virus could not be demonstrated at 0° and might, therefore, be an energy-dependent process. Significant budding activity was also induced at the nonpermissive temperature by incubating cells with 0.25% glutaraldehyde or with antiserum to the major virus envelope glycoprotein, gp70. Anti-gp70 serum may induce budding by promoting aggregation of gp70-containing molecular assemblies and, consequently, association of core components in some transmembrane fashion. Induction of virus assembly with glutaraldehyde might occur as a results of nonspecific cross-linking of membrane proteins. These results suggest that procedures commonly used to minimize ligand-induced redistribution of cell surface molecules, i.e., labeling at low temperatures or after mild aldehyde fixation, may not immobilize certain membrane-associated molecules.     

Immunostaining of intermediate filament proteins in paraffin sections. Evaluation of optimal protease treatment to improve the immunoreactivity

Different protocols of protease (pepsin) treatment were compared in the immunostaining for intermediate filament (IF) proteins in formaldehyde-fixed and paraffin-embedded tissues. Without protease treatment, the immunoreactivity for all IF proteins was poor in such material. Appropriate pepsin pretreatment improved the immunoreactivity of formaldehyde-fixed tissues for all types of IF proteins, except for the 68 K neurofilament protein, which could not be immunostained with the antibody used. The optimal time for the pepsin treatment was varying in different tissues, and too long treatment caused progressive loss of immunostaining. Thus, for instance when demonstrating cytokeratins, renal adenocarcinomas were more sensitive to protease and needed a shorter treatment than other carcinomas. Therefore, a nonoptimal protease treatment protocol may cause false negative results and false cell type selective IF immunostaining. Prolonged fixation made it necessary to prolong the protease treatment. In tissues fixed up to four years in formalin, cytokeratin immunoreactivity could still be restored by a long pepsin treatment (up to 2-3 hours). For most tissues fixed for 24 hours in formaldehyde, an optimal protocol was the following: 0.05% pepsin (2 U/ml) in HCl, pH 1.8, at +37 degrees C for 20-30 minutes. The protease treatment did not produce false positive results. Alcohol-fixed material was good for IF immunostaining without any protease treatment, but such tissue blocks mostly lost the immunoreactivity during long term storage.     

Proliferation in malignant mesothelioma as determined by mitosis counts and immunoreactivity for proliferating cell nuclear antigen (PCNA)

In order to assess its discriminating and prognostic value, we studied immunoreactivity for proliferating nuclear cell antigen (PCNA) in human malignant mesothelioma (31 cases) and in human non-neoplastic mesothelium (33 cases with reactive mesothelium and 20 cases of normal mesothellum) using the murine monoclonal antibody PC 10. We also compared it with mitosis counts expressed as the mitotic volume index (MV index). There were differences between malignant mesothelioma, reactive mesothelium, and normal mesothelium for percentage of PCNA immunoreactive cells (mean ± SD; 27 ± 9, 9·5 ± 5·1, and 3·6 ± 1·6, respectively) and for their MV index (20·3 ± 4·5, 9·4 ± 2·1, and 3·6 ± 0·6, respectively). The median actuarial survival was 10·1 months for patients with less than 25 per cent PCNA immunoreactive cells, 9·4 months for patients with less than 20 mitoses per mm² of tumoural tissue, 5·9 months for patients with more than 25 per cent PCNA immunoreactive cells, and 5·3 months for patients with more than 20 mitoses per mm² of tumoural tissue. Our results suggest that PCNA immunoreactivity is useful in discriminating between neoplastic and non-neoplastic mesothelium and that it may have prognostic value in malignant mesothelioma.     

Immunohistochemical and molecular analysis of p53, MDM2, proliferating cell nuclear antigen and Ki67 in benign and malignant peripheral nerve sheath tumours

A series of 26 malignant peripheral nerve sheath tumours (MPNST) and 24 benign peripheral nerve sheath tumours (BPNST) were analysed immunocytochemically for p53 expression and the cell proliferation markers proliferating cell nuclear antigen (PCNA) and Ki67 (with MIB1). In 23/26 MPNST, 5%–65% of the tumour cell nuclei were immunoreactive for Ki67 with MIB1 while none of the 24 BPNST had nuclear staining exceeding 5%. Greater than 50% nuclear PCNA staining was detected in 25/26 MPNST compared with 8/24 BPNST; 17/26 MPNST showed 5–100% nuclear staining for p53 (13/26>20%), whereas none of the BPNST had nuclear staining exceeding 1%. The Ki67, PCNA and p53 immunostaining results correlated significantly with benign versus malignant (P<0.001, P<0.001 and P<0.005, respectively) as well as mitotic rate (P<0.001, P<0.05 and P<0.05). Ki67 immunostaining results correlated significantly with PCNA and p53, as did p53 with Ki67 and PCNA (P<0.001 in both). Stepwise (logistic regression forward) multivariate analysis of the variable, benign versus malignant, revealed the strongest correlations with PCNA (P=0.007) and Ki67 (P=0.021). Direct confirmation of the presence of p53 protein was obtained by western blot analysis of 3 MPNST and 5 BPNST. Two MPNST, showing 90% and 30% immunoreactivity, were positive for p53, while one MPNST with 5% immunoreactivity and all 5 BPNST were negative. Southern blot analysis performed on the two MPNST with high p53 protein levels revealed no amplification of the MDM2 gene, suggesting that high p53 levels in MPNST are likely to be due to mutation. The results also indicate that PCNA and Ki67 are potentially useful in distinguishing BPNST from MPNST, particularly in problematic cases of cellular schwannoma versus MPNST. The detection of p53 in a large percentage of cells of a plexiform neurofibroma giving rise to MPNST and Ki67 in 5% and 25% of cells of two similar cases suggests that malignant transformation may be detected in some cases by p53 and proliferation markers prior to overt histological evidence of malignancy.