Dr. Werner Bo¨cker Consultation and Reference Center for Gyneco and Breast Pathology Fangdieckstrasse 75a Hamburg Germany [email protected] . Ofner D; Bankfalvi A; Riehemann K; Bier B; Bocker W; Schmid KW, stain method for formalin fixed and paraffin embedded material] Pathologe Quality Control1. Tina Bocker,2Johannes Diermann, Waltraut Friedi, Johannes Gebert, Elke Holinski-Feder,Judith Karner-Hanusch, S.]; and institut fuer Pathologie der Universitaet Regensburg. D .. Manual, Ed. 2.
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PDF | Nonsteroidal anti-inflammatory drugs (NSAIDs) are well-known cancer preventives, which have been largely Josef Rüschoff at Pathologie Nordhessen. PDF | Two monoclonal antibodies, KA 1 and KA 4, raised against human R B Nagle, W Böcker, J R Davis, H W Heid, M Kaufmann, D O Lucas and E D Jarasch February · Verhandlungen der Deutschen Gesellschaft für Pathologie. Igor B. Buchwalow, Werner Böcker. Pages PDF · Background Staining, Autofluorescence and Blocking Steps. Igor B. Buchwalow, Werner Böcker.
Fusion creates healthy mitochondria, whereas fission results in removal of non-functional organelles. Changes in mitochondrial dynamics typify several human diseases. However, the contribution of mitochondrial dynamics to preeclampsia, a hypertensive disorder of pregnancy characterized by placental cell autophagy and death, remains unknown. Increased phosphorylation of DRP1 p-DRP1 in mitochondrial isolates from preeclamptic placentae and transmission electron microscopy corroborated augmented mitochondrial fragmentation in cytotrophoblast cells of PE placentae. Increased fission was accompanied by build-up of ceramides CERs in mitochondria from preeclamptic placentae relative to controls. Treatment of human choriocarcinoma JEG3 cells and primary isolated cytrophoblast cells with CER enhanced mitochondrial fission.
Vietnam materia medica. Hanoi, Ministry of Health, The Indian pharmaceutical codex. Indigenous drugs. British herbal pharmacopoeia, Part 2. London, British Herbal Medicine Association, Iwu MM. Handbook of African medicinal plants. Medicinal plants in Viet Nam. Pharmacognosy, 9th ed. Medicinal plants of India, Vol. Kartnig T. Clinical applications of Centella asiatica L. Phoenix, AZ, Oryx Press, — Farnsworth NR, Bunyapraphatsara N, eds. Thai medicinal plants. Bangkok, Prachachon, Quality control methods for medicinal plant materials.
Geneva, World Health Organization, Deutsches Arzneibuch Methoden der Biologie.
Stuttgart, Deutscher Apotheker Verlag, European pharmacopoeia, 3rd ed. Strasbourg, Council of Europe, Guidelines for predicting dietary intake of pesticide residues, 2nd rev. Determination of asiaticoside contained in sanjinplan. Zhongguo yaoke daxue xuebao, , — The Centella asiatica. Bolletin chimica farmacia, , — Reynolds JEF, ed. Martindale, the extra pharmacopoeia, 30th ed. London, Pharmaceutical Press, Gravel JA.
Oxygen dressings and asiaticoside in the treatment of burns. Laval medicine, , — Bosse JP et al. Clinical study of a new antikeloid agent. Annals of plastic surgery, , — Morisset R et al. Evaluation of the healing activity of Hydrocotyle tincture in the treatment of wounds. Phytotherapy research, , Chaudhuri S et al. Use of common Indian herb Mandukaparni in the treatment of leprosy preliminary report.
Journal of the Indian Medical Association, , — Darnis F et al. Use of a titrated extract of Centella asiatica in chronic hepatic disorders. Semaine hospitaux de Paris, , — Bonte F et al. Influence of asiatic acid, madecassic acid, and asiaticoside on human collagen I synthesis. Planta medica, , — Maquart FX et al.
Stimulation of collagen synthesis in fibroblast cultures by triterpene extracted from Centella asiatica. Connective tissue research, , — May A. The effect of asiaticoside on pig skin in organ culture. European journal of pharmacology, , — Effect of asiaticoside on wound healing in the rat.
Arthroscopically assisted coracoclavicular fixation consisted of a thorough diagnostic arthroscopy through a standard posterior portal including 8 steps of imaging: After entrance into the glenohumeral joint by use of blunt trocar, the first view was the triangle formed by the long biceps tendon, the humeral head and the subscapularis tendon.
The biceps origin was manipulated using a probe in order to detect a SLAP lesion. Hereafter the articular surface of the glenoid and humerus and the medial gleno-humeral ligament MGHL were examined 5th view. The 6th view was on the supraspinatus tendon and the footprint in abduction and external rotation, while the 7th view was used to exclude infraspinatus tendon tears and avulsion of the teres minor or a posterior avulsion of the glenohumeral ligament reversed HAGL.
Finally, the 8th view was to assess the axillary recess for the presence of loose bodies, lesions to the inferior gleno-humeral ligament IGHL and HAGL lesions.
Cartilage injury of the glenoid or the humeral head was evaluated for in all of 8 standard views. Subacromial arthroscopy was not performed in patients of group 1. All patients of group 2 underwent diagnostic arthroscopy at the time of implant removal, which was then performed in the identical manner as described above. In addition, a subacromial arthroscopy was conducted prior to implant removal. Arthroscopic images were blinded retrospectively in order to eliminate interpretational bias by the surgeon or the physical examination.
All images were evaluated independently by two investigators. A third antigen can be detected triple labeling by simply continuing the same protocol with an additional staining sequence appropriate for the third primary antibody. Multiple labeling can increase the time required for staining antigens. If necessary, choose an appropriate stopping point during the staining protocol.
If the staining protocol must be interrupted, it should be after substrate development of the first antigen. However, a danger of potential cross-reactivity still persists.
Applied prior to the use of next primary antibodies, this step serves to remove previously bound primary and link antibodies, leaving only the deposit of chromogen from the previous steps, thus eliminating any potential for cross-reactivity. Guided by the philosophy that if you mix your own reagent solution you know what you have got, the group of Pirici, Mogoanta et al.
Moreover, the glycine SDS elution buffer provided an efficient inhibition of AP and HRP; this might extend its applicability to also inhibiting endogenous enzymatic activity on difficult tissues, instead of separate incubations with hydrogen peroxide or levamisole. The first attempt to perform consecutive immunostaining in the same tissue section with the use of an elution buffer was undertaken as early as 40 years ago by Tramu et al.
Concluding this section, we have to admit that sequential immunoenzymatic double or multiple staining severely increases the time required for staining antigens. Therefore, in our practice we prefer simultaneous immunoenzymatic double staining with primary antibodies noncovalently labeled with different reporter molecules using monovalent Fab fragments as a bridge. Such in-house-made Fab fragment complexes of different primary antibodies of the same species and isotype bearing different labels can be used pairwise to label multiple antigens in the same sample in a single incubation, in the absence of erroneous cross-reactions between different primary and secondary antibody pairs see Sect.
This staining provides blue violet nuclear staining without obscuring antigen-specific chromogen deposition, giving excellent color contrast with most commonly-used peroxidase and alkaline phosphatase substrates in brightfield microscopy see Sect.
Following immunohistochemical staining, wash briefly in distilled water. MHS can be stored for years without losing its potency. Wash in running tap water for 5 10 min.
If the running tap water is not sufficiently alkaline to blue the sections, add a few drops of ammonium hydroxide or a small pinch of calcium hydroxide to about ml of water, then rinse in tap water again. Rinse in distilled water and mount with an aqueous mounting medium, or proceed further with steps 5, 6 and 7.
Clear in two changes of xylene, 5 min each. Mount with xylene-based mounting medium, like Canada balsam or DPX; however, aqueous medium can also be used see Sect.
Convenient ready-to-use nuclear counterstains in blue, green or red colour like Hematoxylin, Methyl Green and Nuclear Fast Red are available from Vector Laboratories http: J Histochem Cytochem 57 6: Scion Publishing, Cambridge Tramu G, Pillez A, Leonardelli J An efficient method of antibody elution for the successive or simultaneous localization of two antigens by immunocytochemistry. Simultaneous immunolocalization of antigens using fluorescent antibodies can be fulfilled both by the direct see Sect.
With the direct method, primary antibodies are labeled with fluorescent dyes, while with the indirect method, primary antibodies are applied as unlabeled antibodies and the visualization is performed with secondary antibodies that are labeled with fluorescent dyes.
Indirect methods for immunofluorescent detection of multiple tissue antigens in their simplest form make use of primary antibodies that are raised in different species and accordingly can be visualized with differently labeled species-specific secondary antibodies see Sect. However, quite often the appropriate combination of primary antibodies from different host species is not available. A general problem relates to the fact that the available primary antibodies may originate only from one species either rabbit or mouse.
When primary antibodies are raised in the same host species, the secondary species-specific antibodies can cross-react with each of the primary antibodies Ino However, if primary antibodies of the same species belong to different IgG isotypes, they can be selectively detected with secondary antibodies directed against the corresponding isotype see Sect. If the primary antibodies are of the same species and of the same IgG isotype, they can be modified via their haptenylation.
Haptenylated primary antibodies are subsequently visualized with the use of secondary antibodies recognizing the corresponding hapten see Sect. The cross-reaction of secondary species-specific antibodies with primary antibodies from the same species is obviously avoided by direct one antibody layer methods. The direct method offers an easy way for simultaneous labeling of a pair or more antigens, even when using primary antibodies from the same species. Recently, a direct technique with primary antibodies that are covalently labeled by different fluorophores was described for a simultaneous detection of up to seven I.
The advantages of directly labeled antibodies are many, including elimination of secondary reagents, lower backgrounds, fewer washes, less hands-on time, and improved data quality. Unfortunately, the desired fluorophore-labeled primary antibodies are not always available. Moreover, this technique suffers from a lower sensitivity compared with indirect methods. For these reasons the majority of researchers rely on indirect two or more antibodies layer methods for their multilabeling requirements.
Bound primary antibodies can accordingly be visualized with differently labeled species-specific secondary antibodies. Primary antibodies to a pair of different antigens e. The same is valid for a mix of secondary antibodies e. Basic protocol for double immunofluorescence staining using primary antibodies raised in two different host species 1 Deparaffinize and rehydrate tissue sections.
When using fluorophore-labeled primary antibodies as in direct immunostaining method one antibody layer , you may skip step 6 with secondary antibodies for indirect immunostaining method two antibodies layers. As stated above, this approach is applicable only when primary antibodies are raised in different species. For double or multiple indirect immunofluorescence staining with primary antibodies raised in the same species, see Sects. This approach has long been used in flow cytometry.
With a broader availability of reliable specific antiIgG subclass antibodies, this approach acquires a growing importance in research and clinical practice. To this end we have elaborated a basic protocol see below for multiple immunolabeling using monoclonal primary antibodies of different IgG isotypes Buchwalow et al. This protocol can be widely applicable, and offers a simple procedure for simultaneous detecting two or more antigens. Basic protocol for multiple immunostaining using monoclonal primary antibodies of different IgG isotypes 1 Deparaffinize and rehydrate tissue sections.
Incubate sections for 60 min at room temperature with a mixture of secondary fluorophore-conjugated antibodies raised against the corresponding IgG isotypes of primary antibodies.
Nuclei were counterstained with DAPI. In its pure form, this approach is applicable to monoclonal primary antibodies belonging to different IgG subclasses. However, combined with haptenylated monoclonal antibodies or with antibodies from other host species, the limits of this technique can be widely extended. Hapten e. Haptenylated primary antibodies can be subsequently visualized with the use of secondary antibodies recognizing the corresponding hapten Fig.
Fluorophorelabeled primary antibodies can be directly visualized in a fluorescent microscope. Although the desired haptenylated primary antibodies are not always available, recent developments in the technology of covalent and noncovalent labeling of antibodies allow the researcher to overcome this problem using his own in-housemade antibody conjugates see Chap.
For convenient protein-labeling procedures, a number of haptens various fluorophores, enzymes, biotin, digoxigenin, etc.
Innova Biosciences http: In our laboratory, we prefer to carry out the noncovalent labeling of primary antibodies ourselves using labeled monovalent Fab fragments see Sect. These protocols can be easily customized depending on the availability of primary antibodies for your research requirements. For instance, you may have at your disposal a pair of monoclonal antibodies of the same IgG isotype, and only one of them is haptenylated.
In this case, you have to carry out the immunostaining in two steps: If the hapten is biotin, you can visualize the biotin label through a fluorophore-conjugated avidin or streptavidin employing the ABC technique see Sect.
Acta Histochem Clin Exp Immunol Antigen detection with the use of antibodies on homologous tissues e. More often than not, antibodies with the required specificities are available in only one species. When working with mouse monoclonal antibodies on mouse tissues, a cheap alternative is to stain with antimouse isotype-specific secondary antibodies, exploiting the relative lower levels of each IgG isotype in the serum and in some cells in the mouse tissue.
Most mouse monoclonal antibodies working on mouse tissue are of IgG1 isotype. Be aware of possible odd crossreactivities e. Important, young laboratory mice raised in sterile, pathogen-free barriers, and unimmunized, have lower endogenous immunoglobulin levels than older animals raised in conventional housing Cattoretti and Qing Unfortunately, this approach does not always help.
There are, however, better ways of avoiding the cross-reactivity with endogenous tissue immunoglobulins.
First, you can use haptenylated primary antibodies, which makes it possible to evade application of secondary anti-IgG antibodies that can bind both to the primary antibody and to irrelevant endogenous tissue immunoglobulins homologous to the primary antibody.
Second, you can preincubate your specimen with unconjugated monovalent Fab fragments, which makes it possible to block endogenous tissue immunoglobulins that are responsible for severe background staining.
Haptenylated primary antibodies can be visualized using secondary antibodies recognizing the corresponding hapten. Primary antibodies haptenylated with a fluorophore can be directly visualized in a fluorescent microscope. Noncovalent labeling of primary antibodies in vitro with a reporter molecule using monovalent Fab fragments that recognize both the Fc and F ab0 2 regions of IgG Brown et al. Simple mixing of labeled monovalent Fab fragments with an unconjugated primary antibody rapidly and quantitatively forms a labeling complex labeled Fab fragment attached to primary antibody.
After absorption of unbound monovalent Fab fragments with excess serum from the same species as the primary antibody, the resultant complexes can be used for immunostaining, avoiding cross-reaction of the secondary antibody with endogenous immunoglobulins when applied to tissue samples homologous to the primary antibody species.
Procedure for generating and using primary mouse antibody Fab fragment complexes on mouse tissues Adapted from Brown et al. Primary antibody to Fab fragment ratios of 1: The working concentration of unlabeled monovalent Fab fragments should not be lower than 0.
This simple procedure makes it possible to block endogenous tissue immunoglobulins with practically complete elimination of background staining Fig. Please note that whole IgG or a divalent F ab0 2 fragment should not be used for blocking, since it has two binding sites. After blocking, some of the binding sites may remain open to capture the primary antibody introduced in a subsequent step, which may result in cross-labeling and thus create higher background.
Procedure for blocking immunoglobulins homologous to primary antibody e. Bring paraffin or frozen sections to water. Perform antigen retrieval if needed. Red color accounts for cardiomyocytes and erythrocytes autofluorescence captured under illumination with a filter excit ing the autofluorescence in red spectrum. Nuclei are counterstained with DAPI blue. Courtesy of Stephanie Grote produce background staining, 3 concentration higher than 0.
In this case, a lower concentration may be used. References 81 Incubate sections with DAB substrate solution for 5 min. Wash with tap water briefly. Counterstain with Hematoxylin if desired. Rinse with tap water. Clear with xylene. Coverslip with permanent mounting medium.
In this case, blocking of the endogenous enzyme activity Step 8 can be avoided. Based apparently on the same principle as described above, suitable but rather expensive kits exploiting monovalent Fab fragments to block endogenous tissue immunoglobulins have also been developed commercially, such as Mouse-onMouse M. TM Kits by Vector Laboratories http: Three Vector1 M.
TM kits are available. These kits use the same blocking technology and biotin-avidin detection format, but offer a choice of using either an enzyme-based or fluorescent-based visualization method.
Eaton Publishing, Natick MA, pp Chapter 10 Probes for Staining Specific Cellular Organelles Many research projects require the localization of certain cellular structures, especially organelles. Specific organelle markers endoplasmic reticulum, golgi apparatus, endosomes, lysosomes, nucleus, plasma membrane, mitochondria, peroxisome and centromer including antibodies to organelle-specific proteins are helpful to locate these organelles by immunocytochemical or histological techniques.
Many of the fluorescent probes designed for selecting organelles are able to permeate or sequester within the cell membrane and therefore are useful in living cells , while others must be installed using monoclonal antibodies with traditional immunocytochemistry techniques http: The website http: The proteins are fused with a fluorescent protein and allow direct visualization of the cell organelles see Chap. Fluorescent nuclear counterstaining is recommended for a better interpretation both of immunostaining and tissue morphology.
Fluorescent nuclear or DNA dyes Table DAPI and Hoechst dyes are quite water-soluble and bind externally to AT-rich base pair clusters in the minor groove of double-stranded DNA, with a dramatic increase in fluorescence intensity at nm and nm respectively.
Both dye classes are the most popular dyes in fluorescence microscopy for use in multicolor fluorescent labeling protocols. Their relatively low-level fluorescence emission does not overwhelm signals from green- or red-fluorescent secondary antibodies or FISH probes.
Propidium iodide PI binds to DNA via intercalation to produce orange-red fluorescence with emission maximum at nm. Nuclear fluorescent counterstaining is carried very rapidly for some specimens not longer than 15 s and should be performed after immunostaining.
Some fluorescent DNA stains can also be used for chromosome counterstaining, for detection of hybridized metaphase or interphase chromosomes in fluorescence in situ hybridization assays or for identifying apoptotic cells in cell populations http: For instance, Vybrant Apoptosis Assay Kit 4 Molecular Probes detects apoptosis on the basis of changes that occur in the permeability of cell membranes.
YO-PRO-1 stain selectively passes through the plasma membranes of apoptotic cells and labels them with moderate green fluorescence. Necrotic cells are stained red-fluorescent with propidium iodide.
These dyes may also be used with ultraviolet trans- or epi-illuminator excitation sources. The monomeric cyanine nucleic acid stains exhibit large degrees of fluorescence enhancement upon binding to DNA or RNA up to 1,fold. Consequently, They are present in all dividing cells of normal and tumor tissues, but absent in resting cells.
Ki nuclear antigen expression is of prognostic importance in a variety of cancers. Ki67 and MIB-1 monoclonal and polyclonal antibodies are directed against different epitopes of the same proliferation-related antigen.
This antibody recognizes native Ki antigen and recombinant fragments of the Ki molecule. PCNA is a 36 kDa molecular weight protein also known as cyclin. PCNA is a useful marker of cells with proliferative potential and for identifying the proliferation status of tumor tissue, which is relevant to prognosis. Protein 53 p53 is a transcription factor that regulates the cell cycle and plays an important role in the control of normal cell proliferation.
The name p53 refers to its apparent molecular mass: The best-described functions of p53 are cell cycle arrest and apoptosis as a response to DNA damage. In most human cancers, the p53 gene is damaged. Monoclonal antibodies directed against epitopes at different domains of p53 protein are highly useful tools to investigate the structure function relationship of wild-type and mutant p53 proteins. Increased p53 expression is a frequent finding in malignant tumors.
The inner and outer nuclear membranes are connected at the nuclear pore sites and enclose a flattened sac 86 10 Probes for Staining Specific Cellular Organelles 10 40 nm wide surrounding the nucleus. The inner membrane of the nuclear envelope lies next to a layer of thin filaments which surrounds the nucleus except at the nuclear pores. The principal component of the nuclear lamina is represented by so-called nuclear lamins. Nuclear lamins form a meshwork that stabilizes the inner membrane of the nuclear envelope.
In lesser concentrations, nuclear lamins are distributed throughout the nucleoplasm. Nuclear lamins are also known as Class V intermediate filaments see Sect.
Nuclear lamins were initially identified as the major components of the nuclear lamina Fawcett Due to their position in the nucleus, lamins were originally proposed to support the nuclear envelope and provide anchorage sites for chromatin. Recently, the nuclear lamins have also been found in the nucleoplasm. Antibodies to nuclear lamins are available commercially from many vendors [see the website: In addition to the lamins, vertebrates express several other lamina-associated proteins including: These multiple functions can be achieved by the transient localization of several hundred proteins within the nucleolar structure.
The abundant nucleolar proteins nucleophosmin B23 and nucleolin C23 have been the subject of numerous studies Ugrinova et al. B23 and C23 are useful markers for tumor diagnosis and prognosis. Their higher expression correlates with cellproliferative status and is associated with poorer disease-specific survival Li et al.
It has been suggested that B23 might be involved in the nucleolar localization of another nuclear protein p Valdez et al. C23 and B23 are argyrophilic proteins targeting to nucleolar organizing regions NORs. They can also be identified by silver staining at low pH. NORs are defined as nucleolar components containing a set of argyrophilic proteins, which are selectively stained by silver methods.
A number of studies carried out in different tumor types demonstrated that malignant cells frequently present a The first silver-staining methods employed for AgNOR protein visualization consisted of two successive phases: In order to control the staining reaction better and avoid non-specific background, Ploton et al.
The Ag NOR solution is then immediately poured over the deparaffinized sections, which are then left in the dark, at room temperature, for 40 min. The silver colloid is washed from the sections with distilled deionized water.
There are a number of other proliferation-associated proteins targeted to the nucleolus, such as protein p and fibrillarin. Protein p was originally identified in the nucleoli of human tumor cells by the use of monoclonal antibodies. Another nucleolar marker, fibrillarin, also known as FBL, is a component of a nucleolar small nuclear ribonucleoprotein snRNP particle.
It is thought to participate in the first step in processing preribosomal r RNA. It is associated with the U3, U8, and U13 small nuclear RNAs and is located in the dense fibrillar component of the nucleolus.
This antibody detects a band at close to 34 kDa in all species tested. Conventional fluorescent stains for mitochondria, such as rhodamine and tetramethylrosamine, are readily sequestered by functioning mitochondria. This characteristic limits their use in experiments in which cells must be treated with aldehyde-based fixatives or other agents that affect the energetic state of the mitochondria.
Because the MitoTracker Orange, MitoTracker Red and MitoTracker Deep Red probes are also retained following permeabilization; the sample retains the fluorescent staining pattern characteristic of live cells during subsequent processing steps for immunocytochemistry, in situ hybridization or electron microscopy.
To label mitochondria, cells are simply incubated in submicromolar concentrations of the MitoTracker probe, which passively diffuses across the plasma membrane and accumulates in active mitochondria. Once the mitochondria are labeled, the cells can be treated with aldehyde-based fixatives to allow further processing of the sample.
MitoTracker probes are cell-permeant mitochondrion-selective dyes that contain a mildly thiolreactive chloromethyl moiety.
The chloromethyl group appears to be responsible for keeping the dye associated with the mitochondria after fixation. In addition, MitoTracker reagents eliminate some of the difficulties of working with pathogenic cells because, once the mitochondria are stained, the cells can be treated with fixatives before the sample is analyzed. The mouse monoclonal antibodies to mitochondria from Abcam http: Fresh cells cultured on microscope slide are recommended, while formalin-fixed paraffin embedded tissue sections can also be suitable.
Use muscle tissue for positive control. Alternatively, mitochondria can be visualized either with specific antibodies raised against mitochondrial proteins, such as porin or pyruvate dehydrogenase, or with antibodies against various subunits of the oxidative phosphorylation complex, for instance, cytochrome oxidase.
Mitochondrial subcellular localization is also possible using vital staining with a cationic dye Janus green B or employing enzyme histochemical staining for peroxidase or cytochromoxidase Lojda et al.
This kit employs a primary antibody Mouse IgG2b directed against This antibody recognizes a protein unique to the Golgi apparatus of most vertebrate species however, it may not work for rat cells and is therefore useful for immunodetection and identification of the Golgi apparatus in most cells. This antibody recognizes a 97 kDa protein called golgin, a member of the granin family of proteins and a peripheral membrane protein localized on the cytoplasmic face of the Golgi apparatus.
This antibody recognizes an epitope located on the microtubule-binding peripheral Golgi membrane 58 kDa protein. In addition to the Golgi apparatus, FTCD is localized to the centrosome, more abundantly around the mother centriole. The centrosome localization of FTCD continues throughout the cell cycle and is not disrupted after Golgi fragmentation induced by colcemid and brefeldin A. FTCD in the centrosome may be associated with polyglutamylated residues of centriole microtubules and may play a role in providing centrioles with glutamate produced by cyclodeaminase domains of FTCD Hagiwara et al.
Therefore it is also useful for studies on the effect of microtubule-perturbing agents on the Golgi apparatus. The Golgi complex and Golgi-derived vesicles may be stained using lectins specific for sugar residues present in this compartment, because one of the primary functions of the Golgi is glycosylation of proteins Virtanen et al.
Clathrin is a major protein component of the cytoplasmic face of intracellular organelles, called coated vesicles and coated pits.
A variety of mono- and polyclonal anti-clathrin antibodies are purchasable from Santa Cruz Biotechnology, inc. Shortly after formation of coated vesicles, the clathrin coat is removed and the vesicles are referred to as endosomes. Endosomes are roughly nm in diameter when fully mature. Antibodies to earlier and late endosomes are available from antibodies-online GmbH, Aachen, Germany http: Early endosomal antigen 1 EEA1 is a kDa membrane-bound protein component specific to the early endosomes and is essential for their fusion with early endocytic vesicles for subsequent redistribution of extracellular compounds to 90 10 Probes for Staining Specific Cellular Organelles alternate destinations.
Extracellular materials trapped in the endocytic vesicles can be either passed into the endosomal compartment or returned to the surface.
Some materials that reach the late endosomes are degraded in lysosomes. Lysosomes are organelles containing digestive enzymes acid hydrolases. They are used for the digestion of macromolecules from phagocytosis, endocytosis, and autophagy. Lysosomal hydrolases can be stained using enzyme histochemical methods Lojda et al. The most useful tools for investigating lysosomal properties with fluorescence microscopy are the LysoTracker and LysoSensor dyes developed by Molecular Probes.
These structurally diverse agents contain heterocyclic and aliphatic nitrogen moieties that modulate transport of the dyes into the lysosomes of living cells for both short-term and long-term studies.
Lysosome-associated membrane proteins, LAMP-1 and LAMP-2 are involved in a variety of functions, including cellular adhesion, and are thought to participate in the process of tumor invasion and metastasis.
LAMP-1 and LAMP-2 proteins are sorted at the trans-Golgi network and transported intracellularly via a pathway that is distinct from the clathrin-coated vesicles.
Their expression is increased in patients with lysosomal storage diseases, such as mucopolysaccharidoses, GM2 gangliosidoses, lipid storage disorders, glycoproteinoses, mucolipidoses, or leukodystrophies.
These diseases are associated with the accumulation of substrates within the cell, impairing metabolism. Caveolae Latin for little caves, singular: They were found in most cell types and are particularly numerous in the continuous endothelium of certain microvascular beds e.
Some cell types, such as neurons, may completely lack caveolae. Many functions are ascribed to caveolae, ranging from clathrinindependent endocytosis and transcytosis of various macromolecules including LDL to signal transduction compartmentalization of certain signaling molecules.
Caveolae are responsible for the regulation of important metabolic pathways including direct interaction with G-protein alpha subunits and functional regulation of their activity. Caveolae may play a critical role in several human diseases such as atherosclerosis, cancer, diabetes, and muscular dystrophies. The principal structural component of caveolae membrane is caveolin, a 21 kDa integral membrane protein.
The caveolin gene family is constituted by three members: CAV1 and CAV2 are located next to each other on chromosome 7 and express colocalizing proteins that form a stable hetero-oligomeric complex. Caveolin-1 and caveolin-2 proteins are most abundantly expressed in endothelial cells, smooth muscle cells, skeletal myoblasts, fibroblasts, and 3T3L1 cells differentiated to adipocytes.
In skeletal muscle cells and cardiac myocytes, A variety of mono- and polyclonal antibodies to all caveolin isoforms are purchasable from Santa Cruz Biotechnology, inc. The cytoskeletal filaments are involved in establishing cell shape, and providing mechanical strength, locomotion, intracellular transport of organelles and chromosome separation in mitosis and meiosis.
The cytoskeleton is made up of three kinds of protein filaments: Actin filaments are the thinnest of the cytoskeletal filaments, and therefore also called microfilaments. Polymerized actin monomers form long, thin fibers of about 8 nm in diameter. There are several types of intermediate filament: Keratins are alpha-type fibrous polypeptides with a diameter of 7 11 nm. They are important components of the cytoskeleton in almost all epithelial cells as well as in some non-epithelial cell types.
In , a new nomenclature Schweizer et al. Keratins are, generally, held to be the most ubiquitous markers of epithelial differentiation, and, so far, 20 distinct types numbered by Moll Moll et al. For further detail about keratin immunostaining see Sect.
Microtubules are straight, hollow cylinders about 25 nm in diameter built by the assembly of dimers of alpha tubulin and beta tubulin. They participate in a wide variety of cell activities. Most involve motion. There are two major groups of microtubule motors: They are commonly organized by the centrosome.
The centrosome is located in the cytoplasm attached to the outside of the nucleus. As mitosis proceeds, microtubules grow out from each centrosome toward the metaphase plate.
The centrosome can be specifically immunostained using anti-gamma-tubulin antibodies that can serve as centrosome marker. The detection, localization and characterization of cytoskeletal proteins are fundamental to the understanding of such cell functions as: Cytoskeleton, Inc.
For successful in vitro research on cytoskeletal components, it is vital to use the right buffers. For example, both tubulin and actin polymerization are highly dependent on the buffer conditions. Diverse antibodies to cytoskeletal and cytoskeleton-associated proteins are also purchasable from Sigma http: Pioneering work on these toxins was done by the Nobel laureate Heinrich Wieland in the s.
The most commonly used member of this toxin family, phalloidin, may be downloadd conjugated to biotin or to a wide variety of fluorescent dyes. Phalloidin selectively binds and stabilizes polymerized, filamentous actin without binding monomeric actin. This property makes phalloidin more attractive than actin-specific antibodies for visualization of filamentous actin.
Phalloidin as a useful tool for investigating the distribution of F-actin in cells using fluorescently labeled phalloidin was first reported by Cooper Using phalloidin conjugated to the fluorophore eosin which acts as the fluorescent tag, Capani et al. In this method, known as fluorescence photooxidation, fluorescent molecules can be utilized to drive the oxidation of diaminobenzidine DAB to create a reaction product that can be rendered electron-dense and detectable also by electron microscopy.
Working solution is prepared immediately before use by diluting 10 mL of the methanolic stock solution into ml PBS or whatever buffer is being used. Staining with biotinylated phalloidin requires the use of approximately a five-times higher concentration of the phalloidin conjugate than when staining with fluorescent phalloidin; additionally a fluorescent or enzyme-conjugated avidin or streptavidin detection reagent must be used.
Since phalloidin is cell-membrane impermeable, cells or tissue specimens must be fixed in buffered formalin before staining. Note that methanol can disrupt actin during the fixation process. Aldehyde fixation renders the plasma membrane permeable enough; therefore we have not found the additional step with detergent permeabilization to be necessary. Phalloidin-stained specimens should be imaged within a few days of staining, as the stain will dissociate into the mounting medium over time and produce a background autofluorescence that may obscure fine detail.
F-actin labeling with fluorescent phalloidins 1. Wash two or more times with PBS. When staining with any of the fluorescent phalloidins, dilute 10 mL methanolic stock solution into ml PBS. Mount sections in antifade medium for fluorescence microscopy. Labeling with fluorescent phalloidins may be combined with immunostaining.
In this case, the phalloidin-staining solution can be applied in a mixture with fluorescently labeled secondary antibodies. Combination of immunostaining with fluorescent phalloidins and fluorescently counterstained nuclei are extremely useful in multiple labeling strategies to locate antigens of interest with specific components of the cell.
Trends Biochem Sci 26 1: J Cell Biol J Cell Sci Am J Anat J Biol Chem Chromosoma Cytometry Carcinogenesis A laboratory manual. Cell A new histodiagnostic marker detected by monoclonal antibodies. Am J Pathol Histochem J J Cell Biol 2: Micron BMC Mol Biol 8: Cancer Res 52 If you do not have an antibody raised against the antigen, which you want to visualize, you might consider adding an epitope tag onto this molecule usually proteins.
This technique can be used to detect proteins that are very difficult to isolate and purify for antibody generation, not to say that epitope tagging can be done at a lower cost than antibody preparation. Moreover, this technique can be used to distinguish between two proteins with similar antigenicity.
Adding an epitope tag onto the molecule is done as follows: A number of methods can be used to introduce the reporter gene, including pronuclear injection, retroviralmediated gene transfer, or gene transfer in embryonic stem cells. The tagged protein will be distributed inside the cell according to the original properties of the protein. For more information on obtaining optimal expression of cloned proteins in mammalian cells see, for instance, Kolodziej and Young and Chen et al.
In most cases, epitope tags are constructed of amino acids. Epitope tags range from 6 to 15 amino acids long and are designed to create a molecular handle for your protein.
Although any short stretch of amino acids known to bind an antibody could become an epitope tag, there are a few that are especially popular. Three examples include: When using such invisible epitope tags, the target proteins can be visualized by immunohistochemical procedures with anti-tag antibodies.
Due to the importance of this technology for protein expression, manufacturers Sigma, Serotec, Abcam and many others offer a wide range of products for the detection, isolation and purification of tagged proteins.