RESUMO
Breast cancer is characterized by cellular and molecular heterogeneity. Several molecular events are involved in controlling malignant cell process. In this sense, the importance of studying multiple cell alterations in this pathology is overriding. A well-identified fact on immune response is that it can vary depend on sex. Steroid hormones and their receptors may regulate different functions and the responses of several subpopulations of the immune system. Few reports are focused on the function of estrogen receptors (ERs) on immune cells and their roles in different breast cancer subtypes. Thus, the aim of this review is to investigate the immune infiltrating tumor microenvironment and prognosis conferred by it in different breast cancer subtypes, discuss the current knowledge and point out the roles of estrogens and its receptors on the infiltrating immune cells, as well as to identify how different immune subsets are modulated after anti-hormonal treatments in breast cancer patients.
Assuntos
Biomarcadores Tumorais/imunologia , Neoplasias da Mama , Estrogênios/imunologia , Proteínas de Neoplasias/imunologia , Receptores de Estrogênio/imunologia , Microambiente Tumoral/imunologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Neoplasias da Mama/fisiopatologia , Feminino , Humanos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologiaRESUMO
INTRODUCTION: Adipose tissue is an important estrogen resource and they are involved in breast cancer development. OBJECTIVE: To establish the relationship between adiposity percentage and the estrogen and progesterone receptors immunoreactive score in Mexican women with breast cancer. METHODS: This is a transversal and analytical study. We identified breast cancer women with cancer histologic diagnosis. We calculated: adiposity percentage and immunoreactive score. We performed correlation analysis between adiposity percentage, body mass index, diabetes mellitus and arterial hypertension, with estrogen and progesterone receptors in breast cancer samples. We perform logistic regression and Odds Ratio estimations. RESULTS: We studied 43 patients with breast cancer and we observed association between adiposity percentage and estrogen and progesterone immnunoreactive score (rP 0.470; p 0.003 and rP 0.328; p 0.042, respectively). The most important risk factor in breast cancer positive to estrogen receptors was obesity (OR 19.1, IC95% 2.1 a 169.1, p 0.008), and previous obesity in breast cancer positive to progesterone receptors (OR 20.7, IC95% 2.3 a 185.9, p 0.007). DISCUSSION: Adiposity percentage is an important risk factor to develop breast cancer positive to hormone receptors related with the risk of breast cancer positive to hormonal receptors.
Introducción: El tejido adiposo es una importante fuente de estrógenos, los cuales se encuentran implicados en el desarrollo de cáncer de mama. Objetivo: Establecer la relación entre el porcentaje de adiposidad y el índice de inmunorreactividad de los receptores a estrógenos y a progesterona en mujeres mexicanas con cáncer de mama. Métodos: Estudio transversal analítico en pacientes con cáncer de mama confirmado con estudio histopatológico. Se estimó el % de adiposidad, y el índice de inmunorreactividad. Se realizó el análisis de correlación entre el porcentaje de adiposidad, el IMC, la presencia de DM2 e hipertensión arterial con la expresión de receptores a estrógeno y progesterona y regresión logística con cálculo de Odds Ratio. Resultados: Se estudiaron 43 pacientes con cáncer de mama y se observó asociación entre el porcentaje de adiposidad y el índice de inmunoreactividad para los RE y RP (rP 0,470; p 0,003 y rP 0,328; p 0,042 respectivamente). El factor de riesgo más importante en cáncer positivo a receptores estrogénicos fue la obesidad (OR 19,1, IC 95% 2,1 a 169,1, p 0,008) y obesidad previa en cáncer positivo a receptores a progesterona (OR 20,7, IC 95% 2,3 a 185,9, p 0,007). Conclusión: El porcentaje de adiposidad es un factor de riesgo importante para desarrollar cáncer de mama positivo a receptores hormonales.
Assuntos
Adiposidade/fisiologia , Neoplasias da Mama/metabolismo , Receptores de Estrogênio/imunologia , Receptores de Progesterona/imunologia , Adulto , Idoso , Composição Corporal/fisiologia , Feminino , Humanos , Imuno-Histoquímica , Modelos Logísticos , México/epidemiologia , Pessoa de Meia-Idade , Razão de Chances , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Fatores de RiscoRESUMO
La clasificación molecular del cáncer de mama ha definido grupos de riesgo y tratamientos diferentes, en este trabajo investigamos como se relacionaron estos grupos. Resultados de 320 pacientes vistas entre enero de 2007 y julio de 2009. En pacientes en las cuales se identifica un cáncer de mama en nuestra institución se realiza una evaluación inmunohistoquímica del tumor, con estos resultados se clasifican los tumores en cuatro subtipos: Luminal A y B, Her2neu y Basal Like. En la evaluación clínico-patológica se consideraron: método diagnóstico, edad, presentación clínica, tamaño del tumor, grado de diferenciación y tratamiento inicial. De 320 pacientes el tumor más frecuente fue Luminal A (60,63 por ciento), seguido por Basal Like (28,75 por ciento) Her2neu (9,06 por ciento) y Luminal B (1,56 por ciento). Los de peor pronóstico como los Basal Like y Her2neu, se consiguió mayor número de tumores palpables y menos diagnóstico subclínico por imágenes, mayor frecuencia en los grupos de menor edad, con mayor tamaño al examen físico y tumores más indiferenciados. Fue necesario iniciar tratamiento de quimioterapia de inducción como primera modalidad terapéutica en un 60,68 por ciento de los Basal Like y 62,06 por ciento de Her2neu, superior al 52,57 por ciento de los tumores Luminal A. La clasificación molecular permite identificar a los tumores con peor pronóstico. Las características histológicas también han identificado grupos de riesgo, pero con connotaciones terapéuticas muy generales. La clasificación molecular determina la posibilidad de suministrar tratamientos de quimioterapia más selectivos.
The molecular classification have defined risk groups and different treatments, the present study explores how these groups were related. 320 patients examinee between January 2007 and July 2009 all patients in which we identify breast cancer in our institution is conducting an assessment of immunohistochemistry according to these results are classified in four subtypes: Luminal A and B, Her2nNeu and Basal Like, also evaluating clinical-pathologic characteristic: Diagnostic method, age, clinical presentation, tumor size, degree of differentiation, initial treatment. In 320 patients the most frequent tumor was Luminal A with a 60.63 percent, followed by Basal Like (28.75 percent) Her2 Neu (9.06 percent) Luminal B (1.56 percent). In relation with worst prognosis of tumors were found more palpable tumors and less subclinical diagnostic by imaging, higher frequency in groups of younger women with larger tumors to physical examination and more undifferentiated. Tumors taking all these considerations needed to initiate treatment of induction chemotherapy as first therapeutic modality in 60.68 percent of the Basal and Like a Her2Neu of 62.06 percent, higher than 52.57 percent of the tumors more frequent Luminal A. The molecular classification of breast cancer let us identified tumors with worst prognosis. The histological features of the tumor have also identified risk groups, but with very broad therapeutic connotations, especially because it allows selection of treatments. On the other hand is only the molecular classification that determines the possibility to provide patients, selective chemotherapy treatments.
Assuntos
Humanos , Feminino , Neoplasias da Mama/classificação , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Receptores de Estrogênio/imunologia , Fenômenos Químicos/métodos , Imuno-Histoquímica/métodos , Técnicas EstereotáxicasRESUMO
BACKGROUND: Novel rabbit monoclonal antibodies (RabMab) for estrogen (ER), progesterone (PR) receptors and HER2 evaluation by immunohistochemistry have recently been commercially released. We compared the RabMab anti-ER, anti-PR and anti-HER2 to mouse monoclonal antibodies (Mab) using tissue microarrays (TMA) of breast carcinomas. METHODS: Two TMA containing breast carcinomas were built. Sections were immunostained using anti-ER and anti-PR, Mab and RabMab. The sections stained for ER and PR were evaluated considering positive those tumors in which more than 1% of the tumor cell nuclei stained moderate or strong. For HER2, the immunostained sections were evaluated using the ASCO/CAP guidelines for HER2. Chromogenic in situ hybridization (CISH) was used as the gold standard for HER2 evaluation. CISH was evaluated using the Zymed HER2 CISH interpretation guidelines. RESULTS: RabMab against ER have similar staining patterns compared to the 6F11 (Mab), but stronger than 1D5 (Mab) from three different suppliers. The RabMab against PR provide stronger and sharper immunohistochemical signals compared to Mab. The detection of HER2 protein overexpression was more prevalent with the polyclonal antibodies and RabMab than with the Mab. These were more specific than the RabMab, which were more sensitive when compared to CISH. CONCLUSION: The novel RabMab against ER and PR showed higher intensity of staining than the Mab. The RabMab against HER2 is more sensitive than Mab, however, Mab presented more specificity than RabMab when compared to CISH for HER2 evaluation of breast carcinomas.
Assuntos
Anticorpos Monoclonais , Neoplasias da Mama/química , Carcinoma Ductal de Mama/química , Receptor ErbB-2/análise , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Animais , Feminino , Humanos , Camundongos , Análise em Microsséries/métodos , Coelhos , Receptor ErbB-2/imunologia , Receptores de Estrogênio/imunologia , Receptores de Progesterona/imunologia , Coloração e Rotulagem , Estatísticas não ParamétricasRESUMO
BACKGROUND: Novel rabbit monoclonal antibodies (RabMab) for estrogen (ER), progesterone (PR) receptors and HER2 evaluation by immunohistochemistry have recently been commercially released. We compared the RabMab anti-ER, anti-PR and anti-HER2 to mouse monoclonal antibodies (Mab) using tissue microarrays (TMA) of breast carcinomas. METHODS: Two TMA containing breast carcinomas were built. Sections were immunostained using anti-ER and anti-PR, Mab and RabMab. The sections stained for ER and PR were evaluated considering positive those tumors in which more than 1 percent of the tumor cell nuclei stained moderate or strong. For HER2, the immunostained sections were evaluated using the ASCO/CAP guidelines for HER2. Chromogenic in situ hybridization (CISH) was used as the gold standard for HER2 evaluation. CISH was evaluated using the Zymed HER2 CISH interpretation guidelines. RESULTS: RabMab against ER have similar staining patterns compared to the 6F11 (Mab), but stronger than 1D5 (Mab) from three different suppliers. The RabMab against PR provide stronger and sharper immunohistochemical signals compared to Mab. The detection of HER2 protein overexpression was more prevalent with the polyclonal antibodies and RabMab than with the Mab. These were more specific than the RabMab, which were more sensitive when compared to CISH. CONCLUSION: The novel RabMab against ER and PR showed higher intensity of staining than the Mab. The RabMab against HER2 is more sensitive than Mab, however, Mab presented more specificity than RabMab when compared to CISH for HER2 evaluation of breast carcinomas.
OBJETIVOS: Novos anticorpos monoclonais de coelho (RabMab) para a avaliação imuno-histoquímica de receptores de estrógeno (RE), progesterona (RP) e HER2 foram lançados comercialmente. Comparamos os RabMab anti-RE, anti-RP e anti-HER2 com os anticorpos monoclonais de camundongo (Mab) utilizando tissue microarrays (TMA) de carcinomas de mama. MÉTODOS: Foram construídos dois TMAs de carcinomas de mama. As secções foram marcadas usando anti-RE, anti-RP e anti-HER2, Mab e RabMab através de imuno-histoquímica. As secções marcadas para RE e RP foram avaliadas considerando positivos aqueles tumores nos quais mais de 1 por cento dos núcleos coraram moderadamente ou forte. Para HER2, as secções foram avaliadas utilizando as recomendações da ASCO/CAP para HER2. Hibridização in situ cromogênica (CISH) foi usada como padrão-ouro para avaliação de HER2. CISH foi avaliado utilizando as recomendações da Zymed. RESULTADOS: Os RabMab anti-RE apresentam intensidade de coloração semelhante ao 6F11 (Mab), porém maior que o 1D5 (Mab) proveniente de três diferentes fabricantes. Os RabMab anti-RP apresentaram sinal imunoistoquímico mais forte e delimitado comparado aos Mab. A detecção da superexpressão da proteína HER2 foi mais prevalente entre os anticorpos policlonais e RabMab, que se mostraram mais sensíveis quando comparados com o CISH. CONCLUSÃO: Os novos RabMab anti-RE e RP proporcionaram maior intensidade de coloração que os Mab. O RabMab anti-HER2 apresentou maior sensibilidade que os Mab, porém os Mab apresentaram maior especificidade quando comparados com o CISH para a avaliação de HER2 em carcinomas de mama.
Assuntos
Animais , Feminino , Humanos , Camundongos , Coelhos , Anticorpos Monoclonais , Neoplasias da Mama/química , Carcinoma Ductal de Mama/química , /análise , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Análise em Microsséries/métodos , /imunologia , Receptores de Estrogênio/imunologia , Receptores de Progesterona/imunologia , Coloração e Rotulagem , Estatísticas não ParamétricasRESUMO
A novel generation of rabbit monoclonal antibodies for estrogen (ER) and progesterone (PR) receptor evaluation in breast cancer by immunohistochemistry has been released recently. We compared the novel RabMab anti-ER and anti-PR antibodies with the mouse monoclonal antibodies using a tissue microarray of breast carcinomas. Two cylinders (2mm diameter) of formalin-fixed, paraffin-embedded tissue were obtained from 24 invasive breast cancers and were immunostained using anti-ER mouse (1D5 and 6F11) and rabbit antibodies (SP1 and B644), and anti-PR mouse (PgR312 and PgR636) and rabbit antibodies (SP2 and B645). The immunohistochemistry was evaluated by considering positive those tumors in which more than 10% of the tumor cell nuclei stained independently on the staining intensity. Our results demonstrated that rabbit antibodies against ER have a similar staining pattern compared to the 6F11, but better than 1D5 from three different suppliers. The rabbit antibodies against PR (SP2 and B645) provide a stronger and sharper immunohistochemical signal compared to mouse antibodies (PgR636 and PgR312). Both ER and PR rabbit antibodies allow a lower cost per test because of higher working dilutions compared to mouse antibodies using the same procedure. The novel rabbit antibodies against ER and PR are highly sensitive for immunohistochemical testing of breast carcinomas.
Assuntos
Anticorpos Monoclonais , Biomarcadores Tumorais/análise , Neoplasias da Mama/metabolismo , Imuno-Histoquímica/métodos , Receptores de Estrogênio/imunologia , Receptores de Progesterona/imunologia , Animais , Especificidade de Anticorpos , Feminino , Humanos , Imuno-Histoquímica/economia , Camundongos , Neoplasias Hormônio-Dependentes/metabolismo , Coelhos , Receptores de Estrogênio/biossíntese , Receptores de Progesterona/biossíntese , Sensibilidade e EspecificidadeRESUMO
Several clinical and experimental studies have demonstrated gender dimorphism in immune and organ responsiveness and in the susceptibility to and morbidity from shock, trauma, and sepsis. In this respect, cell-mediated immune responses have been shown to be depressed in males following trauma-hemorrhage, whereas they were aintained/enhanced in proestrus females. Furthermore, sex hormones have been shown to be responsible for this gender-specific immune response following adverse circulatory conditions. More specifically, studies indicate that androgens produce immunodepression following trauma-hemorrhage in males. In contrast, female sex steroids appear to exhibit immunoprotective properties following trauma and severe blood loss. With regard to the underlying mechanisms, receptors for sex hormones have been identified on various immune cells suggesting direct effects of these hormones on the immune cells. Alternatively, indirect effects of sex hormones, ie, modulation of cardiovascular responses or androgen- and estrogen-synthesizing enzymes, might contribute to gender-specific immune responses. Recent studies indicate that sex hormones, eg, dehydroepiandrosterone (DHEA), also modulate the function of peripheral blood mononuclear cells in surgical patients. Thus, the immunomodulatory properties of sex hormones/receptor antagonists/sex steroid synthesizing enzymes following trauma-hemorrhage suggests novel therapeutic strategies for the treatment of immunodepression in surgical patients.
Assuntos
Hormônios Esteroides Gonadais/imunologia , Sepse/imunologia , Fatores Sexuais , Choque Hemorrágico/imunologia , Ferimentos e Lesões/imunologia , Adjuvantes Imunológicos/uso terapêutico , Antagonistas de Receptores de Andrógenos , Androgênios/imunologia , Circulação Sanguínea , Desidroepiandrosterona/imunologia , Desidroepiandrosterona/uso terapêutico , Suscetibilidade a Doenças , Estrogênios/imunologia , Feminino , Humanos , Imunocompetência , Masculino , Receptores Androgênicos/imunologia , Receptores Androgênicos/uso terapêutico , Receptores de Estrogênio/imunologia , Sepse/tratamento farmacológico , Sepse/fisiopatologia , Choque Hemorrágico/tratamento farmacológico , Choque Hemorrágico/fisiopatologia , Índices de Gravidade do Trauma , Ferimentos e Lesões/tratamento farmacológico , Ferimentos e Lesões/fisiopatologiaRESUMO
Several clinical and experimental studies have demonstrated gender dimorphism in immune and organ responsiveness and in the susceptibility to and morbidity from shock, trauma, and sepsis. In this respect, cell-mediated immune responses have been shown to be depressed in males following trauma-hemorrhage, whereas they were aintained/enhanced in proestrus females. Furthermore, sex hormones have been shown to be responsible for this gender-specific immune response following adverse circulatory conditions. More specifically, studies indicate that androgens produce immunodepression following trauma-hemorrhage in males. In contrast, female sex steroids appear to exhibit immunoprotective properties following trauma and severe blood loss. With regard to the underlying mechanisms, receptors for sex hormones have been identified on various immune cells suggesting direct effects of these hormones on the immune cells. Alternatively, indirect effects of sex hormones, ie, modulation of cardiovascular responses or androgen- and estrogen-synthesizing enzymes, might contribute to gender-specific immune responses. Recent studies indicate that sex hormones, eg, dehydroepiandrosterone (DHEA), also modulate the function of peripheral blood mononuclear cells in surgical patients. Thus, the immunomodulatory properties of sex hormones/receptor antagonists/sex steroid synthesizing enzymes following trauma-hemorrhage suggests novel therapeutic strategies for the treatment of immunodepression in surgical patients.
Uma série de estudos clínicos e experimentais demonstram a existência de dimorfismo sexual das respostas imunológicas e orgânicas, bem como da suscetibilidade e morbidade em relação ao choque, ao trauma e à sepse. Respostas imunes celularmente mediadas apresentam-se deprimidas em machos em resposta ao binômio trauma-hemorragia, mas conservados/enaltecidos em fêmeas em proestro. Adicionalmente demonstra-se que os hormônios sexuais são responsáveis por esta dicomotomia de resposta sexualmente específica, em condições cardiovasculares adversas. Estudos específicos indicam que os andrógenos produzem imunodepressão pós-trauma hemorragia em machos. Em contraste, esteróides sexuais femininos parecem exibir propriedades imunoprotetoras após episódios de trauma com ou sem perda importante de sangue. No terreno dos mecanismos subjacentes, foram identificados receptores para hormônios sexuais em várias células do sistema imunológico, sugerindo a existência de efeitos diretos destes hormônios sobre tais células. Alternativamente, observam efeitos indiretos de hormônios sexuais tais como modulação das respostas cardiovasculares das enzimas sintetizadores de andrógeno e estrógeno, que podem contribuir para as estas respostas sexualmente diferenciadas. Estudos recentes indicam que os hormônios sexuais, como por exemplo a dehidroepiandrosterona também modulam a função de células mononucleares da série branca em pacientes cirúrgicos. Assim, as propriedades imunomodulatórias de hormônios sexuais/antagonistas de receptores/enzimas sintetizadores de esteróides após a ocorrência de trauma ou de hemorragia sugerem o caminho para novas estratégias terapêuticas para o tratamento de imunodepressão em pacientes cirúrgicos.
Assuntos
Humanos , Masculino , Feminino , Hormônios Esteroides Gonadais/imunologia , Caracteres Sexuais , Sepse/imunologia , Choque Hemorrágico/imunologia , Ferimentos e Lesões/imunologia , Adjuvantes Imunológicos/uso terapêutico , Androgênios/imunologia , Circulação Sanguínea , Suscetibilidade a Doenças , Desidroepiandrosterona/imunologia , Desidroepiandrosterona/uso terapêutico , Estrogênios/imunologia , Imunocompetência , Receptores Androgênicos/antagonistas & inibidores , Receptores Androgênicos/imunologia , Receptores Androgênicos/uso terapêutico , Receptores de Estrogênio/imunologia , Sepse/tratamento farmacológico , Sepse/fisiopatologia , Choque Hemorrágico/tratamento farmacológico , Choque Hemorrágico/fisiopatologia , Índices de Gravidade do Trauma , Ferimentos e Lesões/tratamento farmacológico , Ferimentos e Lesões/fisiopatologiaRESUMO
Los receptores esteroidales sexuales del tracto genital de la hembra tienen importancia ya que a través de ellos actúan las hormonas responsables de su desarrollo y de sus cambios morfofuncionales. Dado que en su mecanismo de control uno de los factores a tenerse en cuenta son las posibles diferencias entre las distintas especies, los objetivos de nuestro estudio fueron comparar en dos especies distintas la ovina (O) (n=6) y la canina (P) (n=6) la presencia de receptores de estrógenos (RE), progesterona (RP) y de CBG en ovario, tuba uterina y útero de hembras prepúberes (O:n=3; P:n=·3) y adultas (O:n=3; P:n=3). Se realizaron tinción con H-E e inmunocitoquimica según técnica de Stenberger. Los resultados revelaron: en ambas especies en animales durante el ciclo estral inmunorreactividad (IR) positiva marcada para estrógenos en útero, oviducto y ovario siendo comparativamente los RE más numerosos en oviducto de perra que en oveja. En ambos animales en anestro, la IR para RE fue negativa o leve en útero, no evidenciándose su presencia en otros órganos. Los RP variaron según el estadio del ciclo, no mostrando diferencias destacables entre las especies. En las hembras prepúberes de oveja fueron detectados RE Y RP, mientras que en perras éstos no fueron evidenciados. La CBG mostró IR positiva en el tracto de ambos animales durante el ciclo y fue negativa en prepúberes para ambas especies. Se concluye que existen diferencias, especialmente en hembras prepúberes, donde la presencia de RE y RP es detectable en ovejas no así en perras, siendo además, la presencia de RE comparativamente más marcada en tuba uterina de perra en ciclo que en oveja en el mismo estadio.
The sexual steroid receptors of the genital tract of the female has importance since on them the hormones responsible for their development and morphofuncional changes act. Due that in their control mechanism one of the factors to be kept in mind is the possible differences among the diverse species. The objective of our study was to compare in two different species sheep (O) ( n=6) and canine (P) ( n=6) the presence of receptors of estrogens (RE), progesterone (RP) and the presence of CBG in ovary, oviduct and uterus of female prepúberes (O:n=3; Pn=·3) and mature: (O:n=3; Pn=·3) We use H-E tint and and inmunocito chemistry according to technique of Stenberger. The results revealed: in both species of animals during the estral cycle: Inmunorreactivity (RI) positive marked for estrogens in uterus, oviduct and ovary being comparatively much more numerous RE in dog oviduct that in sheep. In both animals in anestro RE was negative or light in uterus, without evidenced of their presence in other organs. The RP varied according to the stage of the cycle not showing remarkables differences among the species. In the prepuber female sheep RE and RP were detected while in dogs they were not evidenced. The CBG showed positivity in the tract of both animals during the cycle and it was negative in both prepuber species We concludes that differences exist especially in prepuber female where the presence of RE and RP are detectable in sheep and no detectable in dogs, being further the comparatively presence of RE in dog oviduct in cycle more marked that in sheep in the same stage.
Assuntos
Cães , Receptores de Estrogênio/agonistas , Receptores de Estrogênio/imunologia , Receptores de Progesterona/agonistas , Receptores de Progesterona/imunologia , Cães , Imuno-Histoquímica , Imuno-Histoquímica/veterinária , Reprodução/imunologia , OvinosRESUMO
The importance of in situ immunodetection of hormone receptors for therapy planning and prognostic evaluation in patients with breast carcinoma is well established. Sensitive detection methods are of utmost importance, especially in poorly fixed tissues, which are not uncommon in routine pathologic practice. The purpose of the present study is to compare immunoexpression of estrogen receptors in 20 cases of invasive ductal carcinoma using two antibodies, 1D5 and 6F11, and to verify the effect of different antigen retrieval solutions and detection systems. Immunoperoxidase was performed on paraffin sections using 1D5 and 6F11 as primary antibodies. Heat-induced antigen retrieval was performed using citrate buffer (pH 6.0) or Tris-EDTA buffer (pH 8.9). Detection was achieved using the following systems: EnVision, EnVision Plus, and labeled streptavidin-biotin peroxidase complex. Reaction was semiquantified from 0 to 4. There were no differences between the two markers, 1D5 and 6F11, except when 6F11 was used with EnVision and citrate buffer, in which case weaker reactivity was observed. Only in this combination (6F11/EnVision) was EDTA buffer significantly better than citrate. Labeled streptavidin-biotin peroxidase complex presented the best results, followed by EnVision Plus.