Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used on microbiological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, therefore allows visualization of the distribution of the target molecule through the sample; the specific region an antibody recognizes on an antigen is called an epitope. There have been efforts in epitope mapping since many antibodies can bind the same epitope and levels of binding between antibodies that recognize the same epitope can vary. Additionally, the binding of the fluorophore to the antibody itself cannot interfere with the immunological specificity of the antibody or the binding capacity of its antigen. Immunofluorescence is a used example of immunostaining and is a specific example of immunohistochemistry; this technique makes use of fluorophores to visualise the location of the antibodies. Immunofluorescence can be used on tissue sections, cultured cell lines, or individual cells, may be used to analyze the distribution of proteins and small biological and non-biological molecules.
This technique can be used to visualize structures such as intermediate-sized filaments. If the topology of a cell membrane has yet to be determined, epitope insertion into proteins can be used in conjunction with immunofluorescence to determine structures. Immunofluorescence can be used as a "semi-quantitative" method to gain insight into the levels and localization patterns of DNA methylation since it is a more time consuming method than true quantitative methods and there is some subjectivity in the analysis of the levels of methylation. Immunofluorescence can be used in combination with other, non-antibody methods of fluorescent staining, for example, use of DAPI to label DNA. Several microscope designs can be used for analysis of immunofluorescence samples. Various super-resolution microscope designs that are capable of much higher resolution can be used. To make fluorochrome-labeled antibodies, a fluorochrome must be conjugated to the antibody. An antigen can be conjugated to the antibody with a fluorescent probe in a technique called fluorescent antigen technique.
Staining procedures can apply to both fixed antigen in the cytoplasm or to cell surface antigens on living cells, called "membrane immunofluorescence". It is possible to label the complement of the antibody-antigen complex with a fluorescent probe. In addition to the element to which fluorescence probes are attached, there are two general classes of immunofluorescence techniques: primary and secondary; the following descriptions will focus on these classes in terms of conjugated antibodies. There are two classes of immunofluorescence techniques and secondary. Primary immunofluorescence uses a primary antibody, chemically linked to a fluorophore; the primary antibody recognizes the target molecule and binds to a specific region called the epitope. The attached fluorophore can be detected via fluorescent microscopy, depending on the messenger used, will emit a specific wavelength of light once excited. Direct immunofluorescence, although somewhat less common, has notable advantages over the secondary procedure.
The direct attachment of the messenger to the antibody reduces the number of steps in the procedure, saving time and reducing non-specific background signal. This limits the possibility of antibody cross-reactivity and possible mistakes throughout the process. However, some disadvantages do exist in this method. Since the number of fluorescent molecules that can be bound to the primary antibody is limited, direct immunofluorescence is less sensitive than indirect immunofluorescence and may result in false negatives. Direct immunofluorescence requires the use of much more primary antibody, expensive, sometimes running up to $400.00/mL. Secondary immunofluorescence uses two antibodies. Multiple secondary antibodies can bind a single primary antibody; this provides signal amplification by increasing the number of fluorophore molecules per antigen. This protocol is more complex and time-consuming than the primary protocol above, but allows more flexibility because a variety of different secondary antibodies and detection techniques can be used for a given primary antibody.
This protocol is possible because an antibody consists of two parts, a variable region and constant region. It is important to realize that this division is artificial and in reality the antibody molecule is four polypeptide chains: two heavy chains and two light chains. A researcher can generate several primary antibodies that recognize various antigens, but all share the same constant region. All these antibodies may therefore be recognized by a single secondary antibody; this saves the cost of modifying the primary antibodies to directly carry a fluorophore. Different primary antibodies with different constant regions are generated by raising the antibody in different species. For example, a researcher might create primary antibodies in a goat that recognize several antigens, employ dye-coupled rabbit secondary antibodies that recognize
The horse is one of two extant subspecies of Equus ferus. It is an odd-toed ungulate mammal belonging to the taxonomic family Equidae; the horse has evolved over the past 45 to 55 million years from a small multi-toed creature, into the large, single-toed animal of today. Humans began domesticating horses around 4000 BC, their domestication is believed to have been widespread by 3000 BC. Horses in the subspecies caballus are domesticated, although some domesticated populations live in the wild as feral horses; these feral populations are not true wild horses, as this term is used to describe horses that have never been domesticated, such as the endangered Przewalski's horse, a separate subspecies, the only remaining true wild horse. There is an extensive, specialized vocabulary used to describe equine-related concepts, covering everything from anatomy to life stages, colors, breeds and behavior. Horses' anatomy enables them to make use of speed to escape predators and they have a well-developed sense of balance and a strong fight-or-flight response.
Related to this need to flee from predators in the wild is an unusual trait: horses are able to sleep both standing up and lying down, with younger horses tending to sleep more than adults. Female horses, called mares, carry their young for 11 months, a young horse, called a foal, can stand and run shortly following birth. Most domesticated horses begin training in harness between the ages of two and four, they reach full adult development by age five, have an average lifespan of between 25 and 30 years. Horse breeds are loosely divided into three categories based on general temperament: spirited "hot bloods" with speed and endurance. There are more than 300 breeds of horse in the world today, developed for many different uses. Horses and humans interact in a wide variety of sport competitions and non-competitive recreational pursuits, as well as in working activities such as police work, agriculture and therapy. Horses were used in warfare, from which a wide variety of riding and driving techniques developed, using many different styles of equipment and methods of control.
Many products are derived from horses, including meat, hide, hair and pharmaceuticals extracted from the urine of pregnant mares. Humans provide domesticated horses with food and shelter, as well as attention from specialists such as veterinarians and farriers. Specific terms and specialized language are used to describe equine anatomy, different life stages and breeds. Depending on breed and environment, the modern domestic horse has a life expectancy of 25 to 30 years. Uncommonly, a few animals live into their 40s and beyond; the oldest verifiable record was "Old Billy", a 19th-century horse that lived to the age of 62. In modern times, Sugar Puff, listed in Guinness World Records as the world's oldest living pony, died in 2007 at age 56. Regardless of a horse or pony's actual birth date, for most competition purposes a year is added to its age each January 1 of each year in the Northern Hemisphere and each August 1 in the Southern Hemisphere; the exception is in endurance riding, where the minimum age to compete is based on the animal's actual calendar age.
The following terminology is used to describe horses of various ages: Foal: A foal of either sex less than one year old. A nursing foal is sometimes called a suckling and a foal, weaned is called a weanling. Most domesticated foals are weaned at five to seven months of age, although foals can be weaned at four months with no adverse physical effects. Yearling: A horse of either sex, between one and two years old. Colt: A male horse under the age of four. A common terminology error is to call any young horse a "colt", when the term only refers to young male horses. Filly: A female horse under the age of four. Mare: A female horse four years old and older. Stallion: A non-castrated male horse four years old and older; the term "horse" is sometimes used colloquially to refer to a stallion. Gelding: A castrated male horse of any age. In horse racing, these definitions may differ: For example, in the British Isles, Thoroughbred horse racing defines colts and fillies as less than five years old. However, Australian Thoroughbred racing defines fillies as less than four years old.
The height of horses is measured at the highest point of the withers. This point is used because it is a stable point of the anatomy, unlike the head or neck, which move up and down in relation to the body of the horse. In English-speaking countries, the height of horses is stated in units of hands and inches: one hand is equal to 4 inches; the height is expressed as the number of full hands, followed by a point the number of additional inches, ending with the abbreviation "h" or "hh". Thus, a horse described; the size of horses varies by breed, but is influenced by nutrition. Light riding horses range in height from 14 to 16 hands and can weigh from 380 to 550 kilograms. Larger riding horses start at about 15.2 hands and are as tall as 17 hands, weighing from 500 to 600 kilograms. Heavy or draft horses are at least 16 hands (64 inches, 16
Neutrophils are the most abundant type of granulocytes and the most abundant type of white blood cells in most mammals. They form an essential part of the innate immune system, their functions vary in different animals. They are formed from stem cells in the bone marrow and differentiated into subpopulations of neutrophil-killers and neutrophil-cagers, they are short-lived and motile, or mobile, as they can enter parts of tissue where other cells/molecules cannot. Neutrophils may be banded neutrophils, they form part of the polymorphonuclear cells family together with eosinophils. The name neutrophil derives from staining characteristics on hematoxylin and eosin histological or cytological preparations. Whereas basophilic white blood cells stain dark blue and eosinophilic white blood cells stain bright red, neutrophils stain a neutral pink. Neutrophils contain a nucleus divided into 2–5 lobes. Neutrophils are a type of phagocyte and are found in the bloodstream. During the beginning phase of inflammation as a result of bacterial infection, environmental exposure, some cancers, neutrophils are one of the first-responders of inflammatory cells to migrate towards the site of inflammation.
They migrate through the blood vessels through interstitial tissue, following chemical signals such as Interleukin-8, C5a, fMLP, Leukotriene B4 and H2O2 in a process called chemotaxis. They are the predominant cells in pus, accounting for its whitish/yellowish appearance. Neutrophils are recruited to the site of injury within minutes following trauma and are the hallmark of acute inflammation; when adhered to a surface, neutrophil granulocytes have an average diameter of 12–15 micrometers in peripheral blood smears. In suspension, human neutrophils have an average diameter of 8.85 µm. With the eosinophil and the basophil, they form the class of polymorphonuclear cells, named for the nucleus' multilobulated shape; the nucleus has the separate lobes connected by chromatin. The nucleolus disappears as the neutrophil matures, something that happens in only a few other types of nucleated cells. In the cytoplasm, the Golgi apparatus is small and ribosomes are sparse, the rough endoplasmic reticulum is absent.
The cytoplasm contains about 200 granules, of which a third are azurophilic. Neutrophils will show increasing segmentation. A normal neutrophil should have 3–5 segments. Hypersegmentation occurs in some disorders, most notably vitamin B12 deficiency; this is noted in a manual review of the blood smear and is positive when most or all of the neutrophils have 5 or more segments. Neutrophils are the most abundant white blood cells in humans; the stated normal range for human blood counts varies between laboratories, but a neutrophil count of 2.5–7.5 x 109/L is a standard normal range. People of African and Middle Eastern descent may have lower counts. A report may divide neutrophils into segmented bands; when circulating in the bloodstream and inactivated, neutrophils are spherical. Once activated, they change shape and become more amorphous or amoeba-like and can extend pseudopods as they hunt for antigens. Neutrophils have a preference to engulf refined carbohydrates over bacteria. In 1973 Sanchez et al. found that the neutrophil phagocytic capacity to engulf bacteria is affected when simple sugars are digested, that fasting strengthens the neutrophils' phagocytic capacity to engulf bacteria.
However, the digestion of normal starches has no effect. It was concluded that the function, not the number, of phagocytes in engulfing bacteria was altered by the ingestion of sugars. In 2007 researchers at the Whitehead Institute of Biomedical Research found that given a selection of sugars, neutrophils engulf some types of sugar preferentially; the average lifespan of inactivated human neutrophils in the circulation has been reported by different approaches to be between 5 and 90 hours. Upon activation, they marginate and undergo selectin-dependent capture followed by integrin-dependent adhesion in most cases, after which they migrate into tissues, where they survive for 1–2 days. Neutrophils are much more numerous than the longer-lived monocyte/macrophage phagocytes. A pathogen is to first encounter a neutrophil; some experts hypothesize. The short lifetime of neutrophils minimizes propagation of those pathogens that parasitize phagocytes because the more time such parasites spend outside a host cell, the more they will be destroyed by some component of the body's defenses.
Because neutrophil antimicrobial products can damage host tissues, their short life limits damage to the host during inflammation. Neutrophils will be removed after phagocytosis of pathogens by macrophages. PECAM-1 and phosphatidylserine on the cell surface are involved in this process. Neutrophils undergo a process called chemotaxis via amoeboid movement, which allows them to migrate toward sites of infection or inflammation. Cell surface receptors allow neutrophils to detect chemical gr
Immunoglobulin G is a type of antibody. Representing 75% of serum antibodies in humans, IgG is the most common type of antibody found in blood circulation. IgG molecules are released by plasma B cells; each IgG has two antigen binding sites. Antibodies are major components of humoral immunity. IgG is the main type of antibody found in blood and extracellular fluid, allowing it to control infection of body tissues. By binding many kinds of pathogens such as viruses and fungi, IgG protects the body from infection, it does this through several mechanisms: IgG-mediated binding of pathogens causes their immobilization and binding together via agglutination. IgG antibodies are generated following class switching and maturation of the antibody response, thus they participate predominantly in the secondary immune response. IgG is secreted as a monomer, small in size allowing it to perfuse tissues, it is the only antibody isotype that has receptors to facilitate passage through the human placenta, thereby providing protection to the fetus in utero.
Along with IgA secreted in the breast milk, residual IgG absorbed through the placenta provides the neonate with humoral immunity before its own immune system develops. Colostrum contains a high percentage of IgG bovine colostrum. In individuals with prior immunity to a pathogen, IgG appears about 24–48 hours after antigenic stimulation. Therefore, in the first six months of life, the newborn has the same antibodies as the mother and the child can defend itself against all the pathogens that the mother encountered in her life until these antibodies are degraded; this repertoire of immunoglobulins is crucial for the newborns who are sensitive to infections above all for the respiratory and digestive systems. IgG are involved in the regulation of allergic reactions. According to Finkelman, there are two pathways of systemic anaphylaxis: antigens can cause systemic anaphylaxis in mice through classic pathway by cross-linking IgE bound to the mast cell receptor FcεRI, stimulating the release of both histamine and platelet activating factor.
In the alternative pathway antigens form complexes with IgG, which cross-link macrophage receptor FcγRIII and stimulates only PAF release. IgG antibodies can prevent IgE mediated anaphylaxis by intercepting a specific antigen before it binds to mast cell–associated IgE. IgG antibodies block systemic anaphylaxis induced by small quantities of antigen but can mediate systemic anaphylaxis induced by larger quantities. IgG antibodies are large molecules with a molecular weight of about 150 kDa made of four peptide chains, it contains two identical class γ heavy chains of about 50 kDa and two identical light chains of about 25 kDa, thus a tetrameric quaternary structure. The two heavy chains are linked to a light chain each by disulfide bonds; the resulting tetramer has two identical halves. Each end of the fork contains an identical antigen binding site; the various regions and domains of a typical IgG are depicted in the figure to the left. The Fc regions of IgGs bear a conserved N-glycosylation site.
The N-glycans attached to this site are predominantly core-fucosylated diantennary structures of the complex type. In addition, small amounts of these N-glycans bear bisecting GlcNAc and α-2,6-linked sialic acid residues. There are four IgG subclasses in humans, named in order of their abundance in serum. Note: IgG affinity to Fc receptors on phagocytic cells is specific to individual species from which the antibody comes as well as the class; the structure of the hinge regions contributes to the unique biological properties of each of the four IgG classes. Though there is about 95% similarity between their Fc regions, the structure of the hinge regions is different. Given the opposing properties of the IgG subclasses, the fact that the immune response to most antigens includes a mix of all four subclasses, it has been difficult to understand how IgG subclasses can work together to provide protective immunity; the Temporal Model of human IgE and IgG function was proposed. This model suggests; the IgG3, though of low affinity, allows IgG-mediated defences to join IgM-mediated defences in clearing foreign antigens.
Subsequently, higher affinity IgG1 and IgG2 are produced. The relative balance of these subclasses, in any immune complexes that form, helps determine the strength of the inflammatory processes that follow. If antigen persists, high affinity IgG4 is produced, which dampens down inflammation by helping to curtail FcR-mediated processes; the relative ability of different IgG subclasses to fix complement may explain why some anti-donor antibody responses do harm a graft after organ transplantation. In a mouse model of autoantibody mediated anemia using IgG isotype swit
Hematoxylin and eosin stain or haematoxylin and eosin stain is one of the principal stains in histology. It is the most used stain in medical diagnosis and is the gold standard. A combination of hematoxylin and eosin, it produces blues and reds; the staining method involves application of hemalum, a complex formed from aluminium ions and hematein. Hemalum colors nuclei of cells blue, along with a few other objects, such as keratohyalin granules and calcified material, which turns blue when exposed to alkaline water; the nuclear staining is followed by counterstaining with an aqueous or alcoholic solution of eosin Y, which colors eosinophilic structures in various shades of red and orange. The staining of nuclei by hemalum is ordinarily due to binding of the dye-metal complex to DNA, but nuclear staining can be obtained after extraction of DNA from tissue sections; the mechanism is different from that of nuclear staining by basic dyes such as thionine or toluidine blue. Staining by basic dyes occurs only from solutions that are less acidic than hemalum, it is prevented by prior chemical or enzymatic extraction of nucleic acids.
There is evidence to indicate that co-ordinate bonds, similar to those that hold aluminium and hematein together, bind the hemalum complex to DNA and to carboxy groups of proteins in the nuclear chromatin. The eosinophilic structures are composed of intracellular or extracellular protein; the Lewy bodies and Mallory bodies are examples of eosinophilic structures. Most of the cytoplasm is eosinophilic. Red blood cells are stained intensely red; the structures do not have to be basic to be called basophilic and eosinophilic. Other colors, e.g. yellow and brown, can be present in the sample. Some structures do not stain well. Basal laminae need to be stained by PAS stain or some silver stains, if they have to be well visible. Reticular fibers require silver stain. Hydrophobic structures tend to remain clear. Hematoxylin is a dark blue or violet stain, basic/positive, it binds to basophilic substances. DNA/RNA in the nucleus, RNA in ribosomes in the rough endoplasmic reticulum are both acidic because the phosphate backbones of nucleic acids are negatively charged.
These backbones form salts with basic dyes containing positive charges. Therefore, dyes stain them violet. Eosin is a red or pink stain, acidic and negative, it binds to acidophilic substances such as positively charged amino-acid side chains. Most proteins in the cytoplasm of some cells are basic because they are positively charged due to the arginine and lysine amino-acid residues; these form salts with acid dyes containing negative charges, like eosin. Therefore, eosin stains them pink; this includes cytoplasmic filaments in muscle cells, intracellular membranes, extracellular fibers. So, in optical microscopy, one can observe: Nuclei in blue/purple Basophils in purplish red Cytoplasm in red Muscles in dark red Erythrocytes in cherry red Collagen in pale pink Mitochondria in pale pink Papanicolaou stain, another popular staining technique Cytopathology Acid-fast Baker JR Experiments on the action of mordants. 2. Aluminium-haematein. Quart. J. Microsc. Sci. 103: 493–517. Kiernan JA Histological and Histochemical Methods: Theory and Practice.
4th ed. Bloxham, UK: Scion. Lillie RD, Pizzolato P, Donaldson PT Nuclear stains with soluble metachrome mordant lake dyes; the effect of chemical endgroup blocking reactions and the artificial introduction of acid groups into tissues. Histochemistry 49: 23–35. Llewellyn BD Nuclear staining with alum-hematoxylin. Biotech. Histochem. 84: 159–177. Marshall PN, Horobin RW The mechanism of action of "mordant" des – a study using preformed metal complexes. Histochemie 35: 361–371. Puchtler H, Meloan SN, Waldrop FS Application of current chemical concepts to metal-haematein and -brazilein stains. Histochemistry 85: 353–364. SIGMA-ALDRICH H&E Informational Primer Routine Mayer's Hematoxylin and Eosin Stain Hematoxylin & Eosin Staining Protocol Rosen Lab, Department of Molecular and Cellular Biology, Baylor College of Medicine) Step by step protocol
Hemidesmosomes are small stud-like structures found in keratinocytes of the epidermis of skin that attach to the extracellular matrix. They are similar in form to desmosomes when visualized by electron microscopy, desmosomes attach to adjacent cells. Hemidesmosomes are comparable to focal adhesions, as they both attach cells to the extracellular matrix. Instead of desmogleins and desmocollins in the extracellular space, hemidesmosomes utilize integrins. Hemidesmosomes are found in epithelial cells connecting the basal epithelial cells to the lamina lucida, part of the basal lamina. Hemidesmosomes are involved in signaling pathways, such as keratinocyte migration or carcinoma cell intrusion. Hemidesmosomes can be categorized into two types based on their protein constituents. Type 1 hemidesmosomes are found in pseudo-stratified epithelium. Type 1 hemidesmosomes have five main elements: integrin α6β4, plectin in its isoform 1a, i. e. P1a, tetraspanin protein CD151, BPAG1e, or bullous pemphigoid antigen isoform e, BPAG2.
Type 1 hemidesmosomes are found in stratified and pseudostratified epithelial tissue. Type 2 hemidesmosomes contain integrin plectin without the BP antigens. Hemidesmosomes have two membrane-spanning components: Integrin α6β4 and Plectin 1a. Integrin α6β4 operates as a laminin-332 receptor. Integrin α6β4 is composed to two β subunit dimers; the larger β4 subunit has domains that bind to calcium. The α6 subunit binds to extracellular BP180, CD151 and laminin-322; when integrin α6β4 binds to Plectin 1a and BPAG1, it associates with the keratin intermediate filaments in the cytoskeleton. Hemidesmosomes are linked to keratin by plectin isoform 1a from the plakin protein family. Plectin is a 500 kDa protein with a long, rod-like domain and a domain at the end that contains an intermediate filament binding site. BPAG2, or, is a transmembrane protein that exists adjacent to integrins, BPAG2 has domains that bind to plectin, integrin β4 subunit in the cytoplasm and integrin α6 and laminin-332 in the extracellular space.
CD151, a protein of the tetraspanin superfamily, resides on the cell surface of keratinocytes and vascular endothelium. CD151 aids in hemidesmosome formation. BPAG1e is an antigen with multiple isoforms that binds to integrin α6β4, BPAG2 and keratin 5 and 14; the main role of BPAG1e is for hemidesmosome stability. Keeping the basal epidermal keratinocytes attached to the basal lamina is vital for skin homeostasis. Genetic or acquired diseases that cause disruption of hemidesmosome components can lead to skin blistering disorders between different layers of the skin; these are collectively coined epidermolysis bullosa, or EB. Typical symptoms include fragile skin, blister development, erosion from minor physical stress. However, the disease can manifest as erosions on the cornea, gastrointestinal tract, muscular dystrophy and muscular deformity. Mutations in 12 different genes that code for parts of the hemidesmosome have led to epidermolysis bullosa. There are three types of EB: EB simplex, dystrophic EB and junctional EB.
In epidermolysis bullosa simplex, layers of the epidermis separate. EBS is caused by mutations coding for plectin and BPAG1e. With junctional epidermolysis bullosa, layers of the lamina lucida separate; this is caused by mutations in integrin α6β4, laminin 322 and BPAG2. In dystrophic epidermolysis bullosa, the layers of the papillary dermis separate from the anchoring fibrils; this is caused by mutations in the collagen 7 gene. Desmosome Epidermolysis bullosa Focal adhesion
The epidermis is the outermost of the three layers that make up the skin, the inner layers being the dermis and hypodermis. The epidermis layer provides a barrier to infection from environmental pathogens and regulates the amount of water released from the body into the atmosphere through transepidermal water loss; the epidermis is composed of multiple layers of flattened cells that overlie a base layer composed of columnar cells arranged perpendicularly. The rows of cells develop from stem cells in the basal layer. Cellular mechanisms for regulating water and sodium levels are found in all layers of the epidermis; the word epidermis is derived through Latin from Ancient Greek epidermis, itself from Ancient Greek epi, meaning'over, upon' and from Ancient Greek dermis, meaning'skin'. Something related to or part of the epidermis is termed epidermal; the human epidermis is a familiar example of epithelium a stratified squamous epithelium The epidermis consists of keratinocytes, which comprise 90% of its cells, but contains melanocytes, Langerhans cells, Merkel cells, inflammatory cells.
Epidermal thickenings called. Blood capillaries are found beneath the epidermis, are linked to an arteriole and a venule; the epidermis itself has no blood supply and is nourished exclusively by diffused oxygen from the surrounding air. Epidermal cells are interconnected to serve as a tight barrier against the exterior environment; the junctions between the epidermal cells are of the adherens junction type, formed by transmembrane proteins called cadherins. Inside the cell, the cadherins are linked to actin filaments. In immunofluorescence microscopy, the actin filament network appears as a thick border surrounding the cells, although the actin filaments are located inside the cell and run parallel to the cell membrane; because of the proximity of the neighboring cells and tightness of the junctions, the actin immunofluorescence appears as a border between cells. The epidermis is composed depending on the region of skin being considered; those layers in descending order are: cornified layer Composed of 10 to 30 layers of polyhedral, anucleated corneocytes, with the palms and soles having the most layers.
Corneocytes contain a protein envelope underneath the plasma membrane, are filled with water-retaining keratin proteins, attached together through corneodesmosomes and surrounded in the extracellular space by stacked layers of lipids. Most of the barrier functions of the epidermis localize to this layer.clear/translucent layer This narrow layer is found only on the palms and soles. The epidermis of these two areas is known as "thick skin" because with this extra layer, the skin has 5 epidermal layers instead of 4.granular layer Keratinocytes lose their nuclei and their cytoplasm appears granular. Lipids, contained into those keratinocytes within lamellar bodies, are released into the extracellular space through exocytosis to form a lipid barrier; those polar lipids are converted into non-polar lipids and arranged parallel to the cell surface. For example glycosphingolipids become ceramides and phospholipids become free fatty acids.spinous layer Keratinocytes become connected through desmosomes and start produce lamellar bodies, from within the Golgi, enriched in polar lipids, glycosphingolipids, free sterols and catabolic enzymes.
Langerhans cells, immunologically active cells, are located in the middle of this layer.basal/germinal layer. Composed of proliferating and non-proliferating keratinocytes, attached to the basement membrane by hemidesmosomes. Melanocytes are present, connected to numerous keratinocytes in this and other strata through dendrites. Merkel cells are found in the stratum basale with large numbers in touch-sensitive sites such as the fingertips and lips, they are associated with cutaneous nerves and seem to be involved in light touch sensation. The Malpighian layer is both stratum spinosum; the epidermis is separated from its underlying tissue, by a basement membrane. As a stratified squamous epithelium, the epidermis is maintained by cell division within the stratum basale. Differentiating cells delaminate from the basement membrane and are displaced outward through the epidermal layers, undergoing multiple stages of differentiation until, in the stratum corneum, losing their nucleus and fusing to squamous sheets, which are shed from the surface.
Differentiated keratinocytes secrete keratin proteins, which contribute to the formation of an extracellular matrix, an integral part of the skin barrier function. In normal skin, the rate of keratinocyte production equals the rate of loss, taking about two weeks for a cell to journey from the stratum basale to the top of the stratum granulosum, an additional four weeks to cross the stratum corneum; the entire epidermis is replaced by new cell growth over a period of about 48 days. Keratinocyte differentiation throughout the epidermis is in part mediated by a calcium gradient, increasing from the stratum basale until the outer stratum granulosum, where it reaches its maximum, decreasing in the stratum corneum. Calcium concentration in the stratum corneum is low in part because those dry cells are not able to dissolve the ions; this calcium gradient parallels keratinocyte differentiation and as such is considered a key regulator in the formation of the epidermal layers. El