Double immunostaining

Wang, Q. P., Ochiai, H. Nakai, Y. (1992). GABAergic innervation of serotonergic neurons in the dorsal raphe nucleus of the rat studied by electron microscopy double immunostaining. Brain Res. Bull. 29, 943-8. 

Fig. 8.2 Schematic presentation of double immunostaining using secondary antibodies (AB) raised against corresponding IgG isotypes...

Fig. 13.5 Double immunostaining of the nipple epidermis for K5 (pink) and K10 (green). Note that with increasing differentiation, K5 is lost and the cells express K10...

Fig. 13.9 Double immunostaining of squamous cell metaplasia for K14 (green) an K10 (red) in a papilloma of the breast...

Pizzolo, G. and Chilosi, M. (1984) Double immunostaining of lymph node sections by monoclonal antibodies using phycoerythrin labeling and haptenated reagents. Am. J. Clin. Pathol. 82,44-47. 

Hunyady, B Krempels, K., Harta, G. Y and Mezey, E. (1996) Immunohis-tochemical signal amplification by catalyzed reporter deposition and its application in double immunostaining. J. Histochem. Cytochem. 44, 1353-1362. 

MICROWAVE HEAT-ENHANCED DOUBLE IMMUNOSTAINING OF NUCLEAR AND CYTOPLASMIC ANTIGENS... 

Simultaneous, double immunostaining of two antigens in single cells in sections of formalin-fixed and paraffin-embedded archival tissues can be carried out. This is accomplished by using microwave heating to detect otherwise undetectable nuclear antigens, followed by the labeled avidin-biotin (LSAB) procedure and the alkaline phosphatase (APAAP) protocol to detect cytoplasmic or membranous antigens (Bohle et al 1997). 

Horvath TL, Naftolin F, Leranth C (1992b) p-Endorphin innervation of dopamine neurons in the rat hypothalamus a light and electron microscopic double immunostaining study. Endocrinology 737 1547-1555. 

Fast Red gives the most intense (red) reaction product which contrasts well with hematoxylin. For double immunostaining (in combination with POase/DAB) Fast Blue is preferred due to its contrast with oxidized DAB. Naphthol AS-MX (Stage and Avra-meas, 1976) gives comparable results to naphthol AS phosphate. 

Fig. 9. Aspartate aminotransferase immunoreactivity in glutamic acid decarboxylase (GAD)-immunoreactive neuronal processes in the cerebral cortex. sAAT but not mAAT is colocalized with GAD in fine, probably axonal processes (arrows). Rat sections were double-immunostained by incubation with a mixture of anti-sAAT or mAAT rabbit serum and anti-GAD sheep serum, then with biotinylated anti-rabbit IgG donkey antibody, and finally with Texas Red-conjugated avidin and fluorescein-labeled anti-sheep IgG donkey antibody. The photographs in each row were taken at the same site under different excitations. Asterisks in (a) and (o ) indicate the unlabeled cell body of a pyramidal neuron.

Bendayan M, Stephens H (1984) Double immunostaining procedures techniques and applications. In Polak JM, Varndell LM (eds) Immunolabelling for electron microscopy. Elsevier, New York, NY, pp 143-154... 

To localize NT3R immunocytochemically, the oligodendrocytes grown on glass coverslips were washed twice with PBS and fixed with 3 v/v paraformaldel de in PBS pH 7 4 for 10 min at room temperature. They were then incubated with 2 % BSA in PBS for 60 min at room temperature. For double immunostaining the cells were incubated with 2B3-NTR mAb prepared as described by Luo et al. (42) (1 200 dilution in PBS) and... 

In our laboratory, double immunostaining is carried out as follows Immunostain the slides with one of the primary antibodies and detect it with either a fluorescein- or a rho-damine-conjugated secondary antibody, following steps 1-7 (above). Then immunostain the slides with the second primary antibody and detect it with the appropriate secondary antibody (steps 2-8, above). 

To identify cell population undergoing lipid oxidation or protein oxidation, immunodetection of HNE, a marker of lipid peroxidation [25], or dinitrophenol, a marker of protein oxidation [36], can be performed. It can be coupled with a double immunostaining for a blood vessel marker (i.e., CD31, laminin, or RECA-1). 

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