Three-color immunofluorescence histochemistry allowing triple labeling within a single section.

We describe a procedure for simultaneous immunohistochemical localization of three different neuropeptides, neurotransmitters, or neurotransmitter enzymes within one and the same tissue section and present a number of examples of its application within the brain and periphery. Primary antibodies from three different species were bound to three different neurochemical substances within the same section and were then reacted with three appropriate species-specific antisera conjugated with fluorescein, rhodamine/Texas red, or biotin. The biotinylated secondary antiserum was subsequently reacted with diethylaminocoumarin (DAMC) conjugated to avidin. This combination resulted in green, red, and blue fluorescent labeling of each antigen, respectively. Each fluorescent marker was viewed and photographed discretely using appropriate excitation and suppression filter combinations. The method is well suited for analyzing instances of multiple coexistence at both the level of the cell soma and within terminal regions. More broadly, the feasibility of three-color immunofluorescence histochemistry extends the range with which antigen localization can be used to investigate the morphological bases of relationships and interactions between immunohistochemically characterized neuronal elements.

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A simple method for immunofluorescent double staining with primary antisera from the same species.

Journal of Histochemistry & Cytochemistry ◽

10.1177/41.4.8450202 ◽

1993 ◽

Vol 41 (4) ◽

pp. 627-630 ◽

Cited By ~ 33

Author(s):

S Würden ◽

U Homberg

Keyword(s):

Schistocerca Gregaria ◽

Staining Procedure ◽

Double Staining ◽

Double Immunofluorescence ◽

Double Labeling ◽

Simple Method ◽

Fluorescent Markers ◽

Secondary Antiserum ◽

Secondary Antibodies ◽

Texas Red

We have developed a new double immunofluorescence technique by which two neuroactive substances in the same tissue section can be labeled with primary antisera raised in the same species. The optic lobes of the locust Schistocerca gregaria were used as a model system to develop the staining procedure. FMRFamide-immunoreactive neurons were detected by rabbit antisera against FMRFamide and FITC-conjugated secondary antibodies. Antibodies against the second peptide, pigment-dispersing hormone (PDH), also raised in rabbit, were biotinylated and detected via streptavidin-Texas Red. Crossreactivity of the PDH immunoglobulins with the FITC-conjugated secondary antiserum was prevented by pre-incubation with rabbit gamma globulins. The two peptide immunoreactivities could be conveniently observed on the same section with the different fluorescent markers. This double labeling technique with modified antibodies is easily performed and highly useful for co-localization studies with antisera raised in the same species.

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Immunohistochemical localization of orexin/hypocretin-like immunoreactive peptides and melanin-concentrating hormone in the brain and pituitary of medaka

Neuroscience Letters ◽

10.1016/j.neulet.2007.07.043 ◽

2007 ◽

Vol 427 (1) ◽

pp. 16-21 ◽

Cited By ~ 42

Author(s):

Noriko Amiya ◽

Masafumi Amano ◽

Yoshitaka Oka ◽

Masayuki Iigo ◽

Akiyoshi Takahashi ◽

...

Keyword(s):

Immunohistochemical Localization ◽

Melanin Concentrating Hormone ◽

The Brain

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Immunoreactivity of the calbindin D28k in the parahippocampal gyrus of chinchilla

Bulletin of the Veterinary Institute in Pulawy ◽

10.2478/bvip-2013-0067 ◽

2013 ◽

Vol 57 (3) ◽

pp. 387-391

Author(s):

Radosław Szalak ◽

Jadwiga Jaworska-Adamu ◽

Karol Rycerz ◽

Paweł Kulik ◽

Marcin Bartłomiej Arciszewski

Keyword(s):

Brain Area ◽

Neuronal Cell ◽

Immunofluorescence Staining ◽

Parahippocampal Gyrus ◽

Neuronal Cell Bodies ◽

Entorhinal Area ◽

Calbindin D28k ◽

Retzius Cells ◽

Species Specific ◽

The Brain

Abstract Ten adult male chinchillas were used. The localisation of calbindin D28k (CB) was examined with the use of two types of reactions: immunocytochemical peroxidase-antiperoxidase and immunofluorescence staining with a specific monoclonal antibody against CB. Immunocytochemical examination demonstrated the presence of CB-positive neurons in the following layers of all parts the parahippocampal gyrus (PG): marginal, external cellular, middle cellular, and internal cellular, i.e. in entorhinal area, parasubiculum, and presubiculum. Immunofluorescence staining revealed the presence of CB in both Hu C/Dimmunoreactive (IR) neurons and nervous fibers of the PG. CB-IR neuronal cell bodies were moderately numerous (ca. 10% of Hu C/D-IR neurons) and clearly distinguished from the background. Each layer of the brain area consisted of two types of neurons: pyramidal and multiform. Among the second type of neurons, four kinds of morphologically different neuronal subclasses were observed: multipolar, bipolar, round, and Cajal-Retzius cells. It is concluded that the expression of CB in the PG of the chinchilla is species specific and limited to several subclasses of neurons

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The Organization of Human Language Cortex: Special Adaptation or Common Cortical Design?

The Neuroscientist ◽

10.1177/107385849700300116 ◽

1997 ◽

Vol 3 (1) ◽

pp. 61-72 ◽

Cited By ~ 5

Author(s):

Jeffrey J. Hutsler ◽

Michael S. Gazzaniga

Keyword(s):

Time Course ◽

Human Language ◽

Human Communication ◽

Neural Basis ◽

Language Abilities ◽

Language Functions ◽

Evolutionary Forces ◽

Special Adaptation ◽

Species Specific ◽

The Brain

Understanding the neural basis of language is one of the oldest and most difficult pursuits in neuroscience. Despite decades of accumulated data on aphasic subjects with cortical damage, we still know relatively little of how language functions are represented within the neural circuitry of the brain. A major issue of debate is whether language is a species-specific adaptation built into the neocortex, or a by-product of neocortical expansion. Cognitive studies emphasizing the universal nature of language abilities, the consistencies of language structure, and the consistent time course of language development have all indicated that language abilities are innate and must be built into the brain by evolutionary forces. Comparative studies of primates are equivocal since we have little evidence indicating that primate communication is homologous to human language systems. Much of this confusion is related to a lack of information regarding the neural basis of human communication. Recent anatomical data from human brains indicates that left hemisphere regions can have unique types of organization that may be responsible for functional specialization.

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Characterization of the sarcoplasmic reticulum proteins in the thermogenic muscles of fish.

The Journal of Cell Biology ◽

10.1083/jcb.127.5.1275 ◽

1994 ◽

Vol 127 (5) ◽

pp. 1275-1287 ◽

Cited By ~ 33

Author(s):

B A Block ◽

J O'Brien ◽

G Meissner

Keyword(s):

Sarcoplasmic Reticulum ◽

Direct Evidence ◽

Striated Muscle ◽

High Capacity ◽

Muscle Cells ◽

Immunoblot Analysis ◽

Fluorescent Labeling ◽

T Tubules ◽

The Brain

Marlins, sailfish, spearfishes, and swordfish have extraocular muscles that are modified into thermogenic organs beneath the brain. The modified muscle cells, called heater cells, lack organized myofibrils and are densely packed with sarcoplasmic reticulum (SR), transverse (T) tubules, and mitochondria. Thermogenesis in the modified extraocular muscle fibers is hypothesized to be associated with increased energy turnover due to Ca2+ cycling at the SR. In this study, the proteins associated with sequestering and releasing Ca2+ from the SR (ryanodine receptor, Ca2+ ATPase, calsequestrin) of striated muscle cells were characterized in the heater SR using immunoblot and immunofluorescent techniques. Immunoblot analysis with a monoclonal antibody that recognizes both isoforms of nonmammalian RYRs indicates that the fish heater cells express only the alpha RYR isoform. The calcium dependency of [3H]ryanodine binding to the RYR isoform expressed in heater indicates functional identity with the non-mammalian alpha RYR isoform. Fluorescent labeling demonstrates that the RYR is localized in an anastomosing network throughout the heater cell cytoplasm. Measurements of oxalate supported 45Ca2+ uptake, Ca2+ ATPase activity, and [32P]phosphoenzyme formation demonstrate that the SR contains a high capacity for Ca2+ uptake via an ATP dependent enzyme. Immunoblot analysis of calsequestrin revealed a significant amount of the Ca2+ binding protein in the heater cell SR. The present study provides the first direct evidence that the heater SR system contains the proteins necessary for Ca2+ release, re-uptake and sequestration, thus supporting the hypothesis that thermogenesis in the modified muscle cells is achieved via an ATP-dependent cycling of Ca2+ between the SR and cytosolic compartments.

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Immunohistochemical localization and biochemical characterization of ghrelin in the brain and stomach of the frogRana esculenta

The Journal of Comparative Neurology ◽

10.1002/cne.10291 ◽

2002 ◽

Vol 450 (1) ◽

pp. 34-44 ◽

Cited By ~ 22

Author(s):

Ludovic Galas ◽

Nicolas Chartrel ◽

Masayasu Kojima ◽

Kenji Kangawa ◽

Hubert Vaudry

Keyword(s):

Biochemical Characterization ◽

Immunohistochemical Localization ◽

The Brain

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Localization of human pituitary hormones by multiple immunoenzyme staining procedures using monoclonal and polyclonal antibodies.

Journal of Histochemistry & Cytochemistry ◽

10.1177/34.3.2419389 ◽

1986 ◽

Vol 34 (3) ◽

pp. 287-292 ◽

Cited By ~ 24

Author(s):

S Van Noorden ◽

M C Stuart ◽

A Cheung ◽

E F Adams ◽

J M Polak

Keyword(s):

Alkaline Phosphatase ◽

Binding Sites ◽

Polyclonal Antibodies ◽

Single Cells ◽

Pituitary Hormones ◽

Tissue Binding ◽

Human Pituitary ◽

Single Section ◽

End Products ◽

Species Specific

Mouse monoclonal and rabbit polyclonal antibodies to human pituitary hormones were applied together to sections of normal and neoplastic human pituitary tissue. Binding sites were revealed with species-specific immune reagents combined with various enzymes (peroxidase, alkaline phosphatase, and beta-D-galactosidase). The enzymes were developed separately to give differently colored end-products. Where two hormones were present in the same cell, a mixed color was produced. Up to four hormones could be immunostained in a single section. Multiple immunoenzymatic staining has great potential for the analysis of plural antigen production by single cells and relationships between cells producing different antigens.

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