Evaluación de reactividad inmunohistoquímica con método del Complejo Avidina­Biotina (ABC) / Evaluation of immunohistochemical reactivity with Avidin-Biotin Complex (ABC) method

Inmunohistoquímica es toda técnica que permite detectar in situ componentes celulares y extracelulares por medio de anticuerpos específicos, empleando sistemas de detección enzimáticos. Dentro de los métodos inmunohistoquímicos, la técnica del complejo avidina­biotina(ABC) es ampliamente utilizada debido a su alta sensibilidad. El objetivo del presente estudio fueevaluar la reactividad inmunohistoquímica del anticuerpo 4C4.9 para la detección de la proteínaS-100, utilizando el método ABC. Para la evaluación de la reactividad inmunohistoquímica se utilizaron 2 biopsias de piel humana con diagnóstico histopatológico de melanoma maligno nodular ulcerado y nevus melanocítico intradérmico, provenientes del Laboratorio de Investigación en Biotecnología Animal de la Universidad de La Frontera, Temuco, Chile. Se utilizó el Kit VECTASTAIN®como método de detección, la dilución del anticuerpo 4C4.9 fue 1/250 y la temperatura de incubación fue a 4 ºC ó 37 ºC por 18 horas. Para validar la técnica, se realizó un control positivo y otro negativo para 4C4.9. Los resultados de la tinción inmunohistoquímica por el método del complejo ABC mostraron tinción positiva para la proteína S-100, tanto en melanoma maligno nodular ulcerado, como en nevus melanocítico intradérmico, incubados durante 18 horas a 4 ºC ó 37 ºC. Sin embargo, la inmunotinción fue más intensa cuando el anticuerpo primario se incubó a 37 ºC. Para una correcta interpretación de los resultados, es necesario tener en consideración que la reacción antígeno-anticuerpo se ve influenciada por diversos factores, como la concentración del anticuerpo, el tiempo y la temperatura de incubación. En conclusión, nuestros resultados sugieren incubarlas muestras con el primer anticuerpo (4C4.9) en una dilución de 1/250 en agua destilada, incu-bando durante 18 h a 37 ºC. Se recomienda la utilización del anticuerpo 4C4.9 como apoyo al diagnóstico y diagnóstico diferencial.

Immunohistochemistry is anytechnique that can detect cellular and extracellular components in situ by means of specific antibodies,using enzymatic detection systems. Among immunohistochemical methods, the technique ofavidin - biotin complex (ABC) is widely used because of its high sensitivity. The aim of this study was to evaluate the immunohistochemical reactivity of the4C4.9 antibody for detection of S-100 protein using the ABC method. For the evaluation ofimmunohistochemical reactivity 2 biopsies of humanskin were used with histopathological diagnosis ofulcerated malignant melanoma and melanocyticintradermal nevi from the Research Laboratory onAnimal Biotechnology of the Universidad de La Fron-tera, Chile. The Kit VECTASTAIN® was used asdetection method, the dilution the 4C4.9 antibodywas 1/250 and incubation temperature was at 4 °Cor 37 °C for 18 hours. To validate the technique, apositive control and a negative for 4C4.9 was performed. The results of immunohistochemicalstaining by the method of ABC complex showed positive staining for protein S-100 both in ulcerated malignant melanoma and melanocytic intradermalnevi, incubated for 18 hours at 4 °C or 37 °C.However, immunostaining was more intense when the primary antibody was incubated at 37° C. For acorrect interpretation of the results, it is necessary to take into consideration that the antigen-antibody reaction is influenced by various factors such as the concentration of antibody, time and temperature ofincubation. In conclusion, our results suggest incubating the samples with the first antibody (4C4.9)at 1/250 dilution in distilled water, incubating for 18h at 37 ºC. However, immunostaining was moreintense when the primary antibody was incubated at37° C. For a correct interpretation of the results, it isnecessary to take into consideration that antigen-antibody reaction is influenced by various factors suchas the concentration of antibody, time and temperature of incubation. In conclusion, our results suggest incubating the samples with the first antibody(4C4.9) at 1/250 dilution in distilled water, incubating for 18 h at 37 ºC. The use of the antibody 4C4.9 is recommended to support the diagnosis and differential diagnosis.

Effect of human chorionic gonadotropin (hCG) on expression of vascular endothelial growth factor a (VEGF-a) in human mid-secretory endometrial cells in three-dimensional primary culture.

Several lines of evidence suggest that human uterine endometrial cells can bind human chorionic gonadotropin (hCG) which, in turn, influences the physiology of implantation stage endometrium. Vascular endothelial growth factor (VEGF) appears to be a candidate mediator in this process. However, our knowledge about hCG action on VEGF in human endometrial cells is very thin. In the present study, we have examined microscopically hCG binding to dissociated human endometrial cells collected from mid-luteal phase and maintained in three-dimensional primary co-culture on rat-tail collagen type I biomatrix and examined the effect of different concentrations (0, 1, 10, 100 and 1000 IU/ML) of hCG on VEGF expression and secretion by endometrial cells maintained in the above system. We report that both cytokeratin positive epithelial cells as well as vimetin positive stromal cells from human mid luteal phase endometrium could bind hCG and that their number increased (P < 0.01) steadily with time. Administration of hCG enhanced (P < 0.05) immunoreactive VEGF protein expression in dose dependent manner in endometrial cells retrieved from mid-luteal phase of cycle, and co-cultured in a three-dimensional cell culture system, but with no marked change in VEGF secretion. Collectively, it appears that hCG influences VEGF protein synthesis in human midluteal phase endometrial cells, but has little effect on post-translational regulation and secretion. From physiological homeostasis point of view, it is likely that synthesis and secretion of VEGF exhibits a modular and factorial regulation to achieve a fine tuning of this potent vasotropic agent in receptive stage endometrium.

Comparative immunocytochemistry of isolated rat & monkey pancreatic islet cell types.

BACKGROUND & OBJECTIVES: Data on comparative distribution of the islet cell types in the Indian bonnet monkeys and rats are not available. The aim of the present study was to compare the arrangement of the three islet cell types in the native and in isolated rat and Indian bonnet monkey islets by immunocytochemistry. METHODS: Rat islets isolated by chopped tissue collagenase digestion method and islets of monkey isolated by intraductal collagenase digestion method were immunostained by streptavidin-biotin peroxidase method. RESULTS: Immunocytochemical staining of the isolated islets in both the species revealed the presence of three different cell types: insulin secreting B cells, glucagon secreting A cells and somatostatin secreting D cells. The arrangement of islet cell types in the rats and monkeys was similar to that of the intact islets of the native pancreas but were arranged in a definite pattern. In rats the A and D cells were peripherally arranged around the centrally located B cells. In monkeys the B cells occupied the majority of the periphery while the A and D cells were found mostly in the centre. INTERPRETATION & CONCLUSION: The findings of the present study showed that the arrangement of cell types in the islets was not affected by the isolation procedure. The difference in the arrangement of islet cell types in the two species may reflect special functional adaptations.