Subcellular localization staining

In the confocal microscopy experiment, it is recommended to include a negative control. This could be done by incubating cells with phages at 4°C, which should minimize internalization and thus only result in cell surface localization. In addition, endocytosis inhibitors could be used to monitor this event. The subcellular localization could be assessed by co-staining with antibodies that are reactive with different intracellular compartments. For instance, early endosome can be visualized by an EEA1 antibody, whereas late endosomes can be stained by an antibody against the mannose-6-phosphate receptor. 

Histochemical techniques for detecting substrate before and after enzyme treatment are extremely useful in studies on cellular structure. One of the oldest histochemical tests utilized saliva to identify suspected glycogen or starch. More definitive results are obtained when thin sections of a tissue are incubated in a buffered solution of purified amylase and stained for poly-utc-glycols. Material stained by periodic acid-Schiff reagent in the control, but not in the section exposed to amylase, is assumed to be glycogen or starch. Two more of the numerous histochemical techniques associated with localization of substrate are—using hya-luronidase to locate hyaluronic acid and chondroitin 4- and 6-sulfates (179) and using neuraminidases to locate sialomucins (180). By use of electron microscopy in combination with the histochemical technique subcellular localization can be obtained. 

When establishing a new protocol, the antibody dilution can start at 1 10 up to 1 100 usually more diluted antibodies result in less background. Also, sometimes there is a restriction in the quantity of antibody available in the laboratory, so it is best to find the optimal dilution. The time of incubation can be variable also, depending on thickness and permeability of the tissue and subcellular localization of the antigen. The maximum time for room temperature staining is usually 4-6 h and 18-20 h for overnight incubation at 4°C. 

Fig. 8.4 Schematic diagram illustrating methods for quantifying the subcellular distribution of plasmid DNA (pDNA). After the transfection of rhodamine-labeled pDNA, the endosome/lysosome fraction and nuclear fraction was stained with LysoSenser DND-189 and Hoechst 33342, respectively to discriminate the subcellular localization of pDNA. For the data analysis, the pixel areas of each cluster on plasma membrane, S (mem), endosomes/lysosomes, Sj(end/lys), cytosol s, (cyt) and nucleus S (nuc) were separately summed in each XY-plane, and are denoted as S2 j(mem), S2 j(end/lys), S2 j(cyt) and S2 j(nuc), respectively. The values of S2 j(mem), S2 j(end/lys), and S j(nuc) in each X-Y plane were further summed and are denoted as...

Figure 4. Subcellular localization of the photosensitizer BPD-MA. OVCAR-5 cells were incubated in 92 nM BPD-MA for 3 h and 10 nM rhodamine 123, a mitochondrial probe, for 20 min. Imaging was performed using confocal laser scanning microscopy (CLSM). (A) Exclusively mitochondrial green fluorescence of rhodamine 123 (B) red BPD-MA fluorescence (C) overlay of A -i- B, where yellow indicates co-localization (D) DIC transmission image. The colocalization in (C) indicates that BPD-MA localizes to mitochondria, but also stains other subcellular structures.

Fig. 5. Subcellular localization of BARS. (A) To examine the localization of the individual CtBPs, COS7 cells were transfected with BARS (CtBP3/BARS), CtBPl, or CtBP2, and 24 h after transfection they were fixed with 4% paraformaldehyde for 10 min, and double stained with 1 g/ml affinity purified SNl antibody (green) and an anti-tubulin antibody (red). (B) (C) COS7 cells were transfected with BARS, and 24 h after the transfection they were fixed with 4% paraformaldehyde (B), or first permeabilized with SLO, incubated at 37° for 5 min, and fixed with 4% paraformaldehyde (C), for 10 min at room temperature. The cells were double-labeled with 1 iig/ml affinity-purified SNl antibody (red) and a monoclonal anti-giantin antibody (green) the merged images are also shown. The Golgi and plasma membrane localization of BARS are seen after SLO-permeabiUzation, which removes the soluble cytosolic pool of BARS.

Immunofluorescence staining permits detection of protein antigens in situ, in order to investigate the subcellular localization or cellular distribution within a tissue. The cells or tissue sections are fixed and incubated with the specific primary monoclonal antibody. The antigen-primaiy monoclonal antibody complex is bound by a second antibody conjugated to a fluorescent dye, such as rhodamine-p-isothiocyanate or fluorescein isothiocyanate, for detection by fluorescence microscopy. Immunofluorescence staining is described in more detail in Chapter 16. 

This section provides data on specific transcripts and proteins, including which protein is encoded by which transcript, transcript exon structure, protein domains, and subcellular localization. Expression pattern data (including Northern and Western blot data as well as in situ hybridization and antibody staining data) are presented both in a tabulated form using a controlled vocabulary format and in a detailed free-text format. An image-based expression pattern search tool, which can access data captured in the controlled vocabulary format, is being developed. 

Steady state concentrations of adenosine are maintained through the activities of only three enzymes, 5 -nucleotidase (5 -N), adenosine kinase and adenosine deaminase. Adenosine kinase and adenosine deaminase were located mainly in the soluble fractions of rat cerebellar homogenates, whereas 5 -N was present in subcellular fractions (Philips and Newsholme, 1979), mainly in the synaptosomal fraction (Marani, 1977). Adenosine deaminase-immunoreactivity in rat cerebellum was present with one out of five polyclonal sera prepared by Nagy et ah (1988). Staining was present in most Purkinje cells with a variation in intensity. Staining was observed in the Purkinje cell axons and terminals in the cerebellar and vestibular nuclei. The localization of 5 -N will be discussed below. 

There is evidence tliat PrP " in neurons is concenttated in die synaptic region. The protein is axonally transported to nerve terminals (Borchelt et al, 1994) and is enriched in presynaptic membranes obtained by subcellular fractionation (Herms et al, 1999). It has also been localized to synaptic profiles by immunoelectron microscopy (Fournier et al, 1995 Sales et al, 1998). By light microscopic immuno-cytochemistry, PrP " is found primarily in synaptic fields of the olfactory bulb, limbic structures, striatonigral complex, and cerebellar molecular layer, with little staining of neuronal perikarya or fiber pathways (Sales etal, 1998 Herms etal, 1999). These data would suggest a role for PrP in synaptic function, although additional studies are clearly necessary. 

FAAH has been found mainly in microsomal and mitochondrial fractions of rat brain and liver (Deutsch and Chin, 1993 Desarnaud et al., 1995), and of porcine brain (Ueda et al., 1995). Recent studies performed with confocal microscopy, showed that FAAH is localized intracellularly as a vesicular-like staining, that has no association with the plasma membranes and is partially co-localized with the endoplasmic reticulum (Fig. 4.5). These morphological data were corroborated by biochemical assays of FAAH activity in subcellular fractions, showing that AEA hydrolysis was primarily confined to the endomembrane compartment (Oddi et al., 2005). Moreover, by means of reconstituted vesicles derived from purified membrane fractions, it was demonstrated that transport activity is retained by plasma membrane vesicles devoid of FAAH, thereby indicating that AEA hydrolase activity is not necessary for AEA membrane transport. Overall, by means of confocal microscopy, subcellular fractionation, and... 

---