Single-laser three-color immunolabeling of a histological section by laser scanning microscopy: application to senile plaque-related structures in post-mortem human brain tissue.

Abstract

We describe a method for observing three-color immunofluorescence simultaneously in a histological section under a confocal laser scanning microscope. In this study we investigated the spatial relationship of blood vessels, reactive microglia, and amyloid beta-protein (A beta) deposits in post-mortem brain tissue of patients with Alzheimer's disease. HLA-DR was employed as a marker for activated microglia and von Willebrand factor (vWF) as a marker for vascular endothelial cells. HLA-DR was labeled with R-phycoerythrin (R-PE) and vWF with fluorescein isothiocyanate. A beta-protein was immunostained with the tandem conjugate of R-PE and cyanin 5. Three images were obtained serially by scanning a tissue section with a 488-nm laser beam in combination with an appropriate emission filter for each fluorochrome. Overlaying of these three images permitted simultaneous observation of three distinct structures: activated microglia, blood vessels, and A beta deposits. This technique provides an improved way to study the localization of three different antigens in a single tissue section.

Three-dimensional immunofluorescent double labelling using polyclonal antibodies derived from the same species: enterocytic colocalization of chylomicrons with Golgi apparatus

“…However, these two options for double immunolabelling are often not possible, because commercially available polyclonal antibodies are commonly raised in the same species, or monoclonal antibodies are either the entire IgG or the cross-reactive isoforms of the immunoglobulin. One approach for avoiding cross-reactivity and false-positive signalling is to diVerentially conjugate primary antibodies with labels such as enzymes, biotin or Xuorochromes, prior to their application on tissue (Uchihara et al 1995;Tsurui et al 2000). The latter method generally provides reliable detection of two or more antigens; however, the sensitivity of preconjugation is generally lower compared to when primary antibodies are applied and detected thereafter with labelled secondary antibodies.…”

“…The latter method generally provides reliable detection of two or more antigens; however, the sensitivity of preconjugation is generally lower compared to when primary antibodies are applied and detected thereafter with labelled secondary antibodies. Furthermore, preconjugation typically requires larger quantities of antibodies; the procedure can be rather complex and not always successful (Uchihara et al 1995).…”

Dual enhancement of triple immunofluorescence using two antibodies from the same species

“…Therefore, double labeling with two primary antibodies from the same species and class, for example, two mouse monoclonal IgGs as we demonstrated in this study, may be of great help. Direct conjugation of biotin to one of the primary antibodies may be one of the methods of choice for this purpose, but the procedure is cumbersome and requires a relatively large amount of the primary antibody (Uchihara et al, 1995). Hunyaday et al (1996) was the first who utilized CARD amplification in this way for double fluorolabeling to obtain a good separation from non-amplified signal.…”

Martentoxin, a large-conductance Ca2+-activated K+ channel inhibitor, attenuated TNF-α-induced nitric oxide release by human umbilical vein endothelial cells

“…The cells were cultured in M199 medium containing 10% FBS, 100 IU/mL penicillin and 100 µg/mL streptomycin at 37°C in a fully humidified atmosphere containing 5% CO 2 . The anti-von Willebrand factor antibody was used to label blood vessels[15]. After one week of culture, the HUVECs were identified by immunofluorescence staining with anti-von Willebrand factor antibody[16],[17].…”