Fluorescence Counter-Staining of Cell Nuclear DNA for Multi-Color Laser Confocal Microscopy.

Suzuki, Takeshi; Matsuzaki, Toshiyuki; Takata, Kuniaki

doi:10.1267/ahc.31.297

Cited by 17 publications

(8 citation statements)

References 24 publications

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“…Afterwards, the sections were rinsed 4 ϫ 30 min in TSPB and then incubated for 4 h in a mixture of two secondary antibodies [goat anti-mouse labeled with CY3 (Jackson Immunoresearch) and goat anti-rabbit labeled with Alexa 488 (Molecular Probes)] both diluted 1:150 in TSPB. After rinsing in SPB (4 ϫ 30 min), the sections were incubated for 20 min in Hoechst 33258 (Sigma) diluted 1:100 in SPB from a stock solution of 1 mg/mL and TOPRO-3 (Molecular Probes) diluted 1:2000 (from a stock solution of 1 mM in DMSO) to counterstain all nuclei for epifluorescence or confocal microscopy (Suzuki et al, 1998). After a final rinse in SPB, the sections were coverslipped in (1:1) glycerol/SPB containing 5% DABCO {diazabicyclol[2.2.2]octane; Sigma} to prevent bleaching of the TOPRO-3 fluorescence.…”

Section: Immunocytochemistrymentioning

confidence: 99%

Neuronal differentiation and long‐term survival of newly generated cells in the olfactory midbrain of the adult spiny lobster, Panulirus argus

Schmidt

2001

J. Neurobiol.

103

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The fate of continuously generated cells in the soma clusters of the olfactory midbrain of adult spiny lobsters, Panulirus argus, was investigated by in vivo pulse-chase experiments with the proliferation marker 5-bromo-2'-deoxyuridine (BrdU) combined with immunostainings for neuropeptides of mature neurons. A BrdU injection after a survival time (ST) of 14 h labeled about 100 nuclei in the lateral soma clusters (LC), comprised of projection neurons, and about 30 nuclei in the medial soma clusters (MC), comprised of local interneurons. The BrdU-positive nuclei were confined to small regions at the inside of these clusters, which also contain nuclei in different phases of mitosis and thus represent proliferative zones. After STs of 2 weeks or 3 months, the number of BrdU-positive nuclei was doubled, indicating a mitosis of all originally labeled cells. Dependent on ST, the BrdU-positive nuclei were translocated from the proliferative zones towards the outside of the clusters, where somata of mature neurons reside. Immunostainings with antibodies to the neuropeptides FMRFamide and substance P, both of which label a large portion of somata in the MC and a pair of giant neurons projecting into the LC, revealed that in both clusters the proliferative zones are surrounded by, but are themselves devoid of, labeling. In the MC, some BrdU-positive somata were double-labeled by the FMRFamide antibody after an ST of 3 months, and by the substance P antibody after STs of 6 and 11/14 months, but not after 3 months. In the LC, BrdU-positive somata after an ST of 3 months partially and after 6 and 11/14 months widely overlapped with the arborizations of the giant neurons, indicating the establishment of synaptic input. The experiments show that cells generated in proliferative zones in the LC and MC of adult spiny lobsters after a final mitosis differentiate into neurons within months, survive for at least 1 year, and are integrated into the circuitry of the olfactory midbrain. A new hypothesis about the mechanism of adult neurogenesis in the central olfactory pathway of decapod crustaceans is developed, linking it to neurogenesis during embryonic and larval development.

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Section: Immunocytochemistrymentioning

confidence: 99%

Neuronal differentiation and long‐term survival of newly generated cells in the olfactory midbrain of the adult spiny lobster, Panulirus argus

Schmidt

2001

J. Neurobiol.

103

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“…The labeling pattern is the same as in a. Bars 100 µm for laser confocal microscopy (Matsuzaki et al 1997;Suzuki et al 1998) was included in the secondary antibody solution. To check the specificity of the immunostaining, we performed the following controls: (a) incubation with preimmune rabbit serum (1/500 dilution) instead of anti-AQP5 antibody; (b) incubation with normal rabbit IgG (1.5 µg/ml; Jackson Immunoresearch) instead of anti-AQP5 antibody; (c) incubation with normal rat serum (1/500 dilution; Bethyl Laboratories, Montgomery, TX) instead of anti-occludin antibody; (d) adsorption controls by preincubating the anti-AQP5 antibody (1.5 µg/ml) with COOH-terminus peptide of AQP5 protein (20 µg/ml) used as immunogen; and (e) adsorption controls by preincubating the anti-AQP5 antibody (1.5 µg/ml) with a mixture of COOH-terminus peptides of AQP1, 2, 3, and 4 proteins (20 µg/ml each).…”

Section: Fig 2a-jmentioning

confidence: 99%

Aquaporin-5 (AQP5), a water channel protein, in the rat salivary and lacrimal glands: immunolocalization and effect of secretory stimulation

Matsuzaki

Suzuki

Koyama

et al. 1999

Cell and Tissue Research

Self Cite

172

143

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Aquaporin-5 (AQP5) is a water channel protein and is considered to play an important role in water movement across the plasma membrane. We raised anti-AQP5 antibody and examined the localization of AQP5 protein in rat salivary and lacrimal glands by immunofluorescence microscopy. AQP5 was found in secretory acinar cells of submandibular, parotid, and sublingual glands, where it was restricted to apical membranes including intercellular secretory canaliculi. In the submandibular gland, abundant AQP5 was also found additionally at the apical membrane of intercalated duct cells. Upon stimulation by isoproterenol, apical staining for AQP5 in parotid acinar cells tended to appear as clusters of dots. These results suggest that AQP5 is one of the candidate molecules responsible for the water movement in the salivary glands.

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“…[24,25] However, these fluorophores have several limitations that restrict their utility in applications such as cell proliferation, stem-cell differentiation, and realtime microscopy experiments lasting several hours. [22,[26][27][28][29][30][31] Photobleaching of the chromophore limits the ability of investigators to observe their specimens repeatedly and over long periods.…”

mentioning

confidence: 99%

Single‐Walled Carbon Nanotube Spectroscopy in Live Cells: Towards Long‐Term Labels and Optical Sensors

Heller¹,

Baik²,

Eurell³

et al. 2005

Advanced Materials

515

451

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Fluorescence Counter-Staining of Cell Nuclear DNA for Multi-Color Laser Confocal Microscopy.

Cited by 17 publications

References 24 publications

Neuronal differentiation and long‐term survival of newly generated cells in the olfactory midbrain of the adult spiny lobster, Panulirus argus

Neuronal differentiation and long‐term survival of newly generated cells in the olfactory midbrain of the adult spiny lobster, Panulirus argus

Aquaporin-5 (AQP5), a water channel protein, in the rat salivary and lacrimal glands: immunolocalization and effect of secretory stimulation

Single‐Walled Carbon Nanotube Spectroscopy in Live Cells: Towards Long‐Term Labels and Optical Sensors

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