Molecular Morphology of Pituitary Cells, from Conventional Immunohistochemistry to Fluorescein Imaging

Abstract

In situ hybridization (ISH) at the electron microscopic (EM) level is essential for elucidating the intracellular distribution and role of mRNA in protein synthesis. EM-ISH is considered to be an important tool for clarifying the intracellular localization of mRNA and the exact site of pituitary hormone synthesis on the rough endoplasmic reticulum. A combined ISH and immunohistochemistry (IHC) under EM (EM-ISH&IHC) approach has sufficient ultrastructural resolution, and provides two-dimensional images of the subcellular localization of pituitary hormone and its mRNA in a pituitary cell. The advantages of semiconductor nanocrystals (quantum dots, Qdots) and confocal laser scanning microscopy (CLSM) enable us to obtain three-dimensional images of the subcellular localization of pituitary hormone and its mRNA. Both EM-ISH&IHC and ISH & IHC using Qdots and CLSM are useful for understanding the relationships between protein and mRNA simultaneously in two or three dimensions. CLSM observation of rab3B and SNARE proteins such as SNAP-25 and syntaxin has revealed that both rab3B and SNARE system proteins play important roles and work together as the exocytotic machinery in anterior pituitary cells. Another important issue is the intracellular transport and secretion of pituitary hormone. We have developed an experimental pituitary cell line, GH3 cell, which has growth hormone (GH) linked to enhanced yellow fluorescein protein (EYFP). This stable GH3 cell secretes GH linked to EYFP upon stimulation by Ca2+ influx or Ca2+ release from storage. This GH3 cell line is useful for the real-time visualization of the intracellular transport and secretion of GH. These three methods from conventional immunohistochemistry and fluorescein imaging allow us to consecutively visualize the process of transcription, translation, transport and secretion of anterior pituitary hormone.

2‐Sulfanylhydroquinone Dimer as a Switchable Fluorescent Dye

“…2019, 25,14081 -14088 www.chemeurj.org 2019 Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim 6H), 6.78 (s, 2H), 6.67 (s, 2H), 3.86 (s, 6H), 1.97 ppm (s, 6H); 13 CNMR (126 MHz, CDCl 3 ) d = 169.6, 153. 9, 141.8, 133.2, 132.5, 129.6, 128.7, 128.2, 126.8, 123.4, 112.5, 56.5, 20.8 Preparation of acetylisopropyldimethyl 2-phenylthiohydroquinone dimer 6:2 ,10-dimethoxy-6,6-dimethyl-3,9-bis(phenylthiol)dibenzo-[d,f] [1,3]dioxepine (27.6 mg, 0.0550 mmol) was dissolved in CH 2 Cl 2 (5 mL) and heated to reflux. DIBAL-H (1.5 m toluene solution, 0.30 mL, 0.45 mmol) was added to the hot solution, and the reaction mixture was heated for 3.5 ha tthe same temperature.…”

“…[1] To date, many fluorophores have been devised and used for these purposes. Such fluorophores include derivatives of coumarin, [2] fluorescein, [3] BODIPY, [4] rhodamine, [5] phenoxazine, [6] acridinone [7] and cyanine. [8] When thesem olecules are attached to ab ioactivem olecule, they serve as useful tools for tracing how bioactive molecules are introduced and behave in al iving cell.…”

5-Bromo-4′,5′-bis(dimethylamino)fluorescein: Synthesis and Photophysical Studies

“… 1 The p K a values were calculated using the DATAN program. 2 Reported p K a values [ 3 , 6 ]. 3 Not detectable.…”

“…Fluorescein is one of the most widely used fluorophores in the chemical, biochemical, and medicinal fields due to its ease of synthesis and brilliant green fluorescence [ 1 , 2 , 3 , 4 , 5 , 6 ]. Fluorescein shows complex protolytic equilibria in aqueous solution.…”

Semiconductor Quantum Dots for Biomedicial Applications

“…The main advantage of QDs imaging is that it is non-ionizing and less hazardous [ 108 ]. In recent years, several groups have used QD probes for fluorescence immunostaining of fixed cells and tissue specimens [ 109 – 113 ]. QD-based immunohistochemistry (IHC) can improve both diagnostic sensitivity and specificity.…”