Identity of Holmes Positive Bodies with Electron Microscopically Demonstrable Nucleolus-Like Bodies in Neuronal Cytoplasm

By light microscopic observation of mouse brain stained by Holmes' silver method deeply stained cytoplasmic inclusion bodies were seen in almost all nerve cells of the locus coeruleus. Electron microscopy of tissue samples from floating Vibratome sections stained by Holmes' silver method demonstrated that the nucleolus-like bodies in the cytoplasm were densely impregnated with gold particles. Hence, it was confirmed that the cytoplasmic inclusion bodies of paraffin sections stained by Holmes' method are identical to the so-called nucleolus-like bodies seen in electron microscopic studies.     

A parvo-like virus in cultured redclaw crayfish Cherax quadricarinatus from Queensland,Australia

In the summer of 1999/2000, an epizootic occurred in cultured juvenile redclaw crayfish Cherax quadricarinatus on one commercial crayfish farm in northern Queensland, Australia. Mortalities occurred over 4 wk, with up to 96% cumulative mortalities in 2 earthen ponds stocked with juveniles. The crayfish were weak, anorexic and lethargic. A transmission trial was conducted, using filtered, cell-free extract prepared from infected crayfish as inoculum. The disease was reproduced, with on-going mortalities occurring in inoculated crayfish over 55 d. Experimentally inoculated crayfish showed gross signs of malaise, anorexia and disorientation before dying. Two types of intranuclear inclusion bodies (INIBs) were seen in tissues of endodermal, ectodermal and mesodermal origin by light microscopy with haematoxylin and eosin (H&E) stained sections. 'Early'-stage INIBs were eosinophilic, rounded and located centrally within slightly enlarged nuclei while 'late'-stage INIBs were well-rounded and deeply basophilic. The gills, cuticular epithelium and epithelial cells of the foregut, midgut and hindgut were the most heavily infected tissues. By transmission electron microscopy, virions with an average diameter of 19.5 nm were seen within electron-dense granular inclusion bodies within enlarged nuclei of both naturally and experimentally infected crayfish. The size of the virions and cytopathology are consistent with characteristics of viruses in the Family Parvoviridae. This is the first reported case of mass mortality caused by a parvo-like virus infection in C. quadricarinatus.     

Luna stain, an improved selective stain for detection of microsporidian spores in histologic sections

Microsporidia in histologic sections are most often diagnosed by observing spores in host tissues. Spores are easy to identify if they occur in large aggregates or xenomas when sections are stained with hematoxylin and eosin (H&E). However, individual spores are not frequently detected in host tissues with conventional H&E staining, particularly if spores are scattered within the tissues, areas of inflammation, or small spores in nuclei (i.e. Nucleospora salmonis). Hence, a variety of selective stains that enhance visualization of spores is recommended. We discovered that the Luna stain, used to highlight eosinophils, red blood cells, and chitin in arthropods and other invertebrates, also stains spores of Pseudoloma neurophilia. We compared this stain to the Gram, Fite's acid fast, Giemsa, and H&E stains on 8 aquatic microsporidian organisms that were readily available in our 2 laboratories: Loma salmonae, Glugea anomala, Pseudoloma neurophilia, Pleistophora hyphessobryconis, Pleistophora vermiformis, Glugea sp., Steinhausia mytilovum, and an unidentified microsporidian from UK mitten crabs Eriocheir sinensis. Based on tinctorial properties and background staining, the Luna stain performed better for detection of 6 of the 8 microsporidia. Gram stain was superior for the 2 microsporidia from invertebrates: S. mytilovum and the unidentified microsporidian from E. sinensis.     

Grayscale representation of infrared microscopy images by extended multiplicative signal correction for registration with histological images

Fourier‐transform infrared (FTIR) microspectroscopy is rounding the corner to become a label‐free routine method for cancer diagnosis. In order to build infrared‐spectral based classifiers, infrared images need to be registered with Hematoxylin and Eosin (H&E) stained histological images. While FTIR images have a deep spectral domain with thousands of channels carrying chemical and scatter information, the H&E images have only three color channels for each pixel and carry mainly morphological information. Therefore, image representations of infrared images are needed that match the morphological information in H&E images. In this paper, we propose a novel approach for representation of FTIR images based on extended multiplicative signal correction highlighting morphological features that showed to correlate well with morphological information in H&E images. Based on the obtained representations, we developed a strategy for global‐to‐local image registration for FTIR images and H&E stained histological images of parallel tissue sections.     

Ex-vivo fluorescence confocal microscopy with digital staining for characterizing basal cell carcinoma on frozen sections: A comparison with histology

Ex-vivo fluorescence confocal microscopy (FCM) has been used on fresh tissue, but there is little experience on frozen sections. We evaluated the applicability of FCM on frozen sections of basal cell carcinomas (BCCs), stained with acridine orange and digitally colored to simulate hematoxylin and eosin (H&E) dyes. We compared our diagnostic accuracy in detecting and subtyping BCCs with FCM to our gold standard (H&E stained frozen sections used in 3D horizontal micrographic surgery). Fourty-six primary BCCs were analyzed for free margins as well as histological subtype with all FCM modes and conventional H&E staining. Adnexa, artifacts and diagnostic confidence were evaluated. Free margins were identified with a sensitivity and specificity of 92% and 91%. Concordance for tumor subtype was 88%. FCM may be used on both fresh tissue and frozen samples, although with reduced performance and different artifacts. The device is useful for the intraoperative diagnosis, subtyping and margin-mapping of BCCs.      

Occurrence of lipofuscin pigment granules and "dense microspheres" in the spinal cord of young cats treated with beta,beta'-iminodipropionitrile (IDPN)

Spinal cords of cats treated with the neurotoxic compound beta,beta'-iminodipropionitrile (IDPN) were observed to contain rounded homogeneous bodies, 1-12 microns in diameter, termed "dense microspheres" (DMS). These bodies, absent in control animals, were consistently found only in the ventral horns. No relationship with blood vessels was evident. When stained with PAS and a modified von Kossa's silver nitrate technique, DMS remained negative, showing only very slight metachromasia in some toluidine blue-stained sections. They were consistently acidophilic as evidenced by destaining and differentiation investigations. DMS were observed more frequently in the proximity of nerve cell bodies or closely adjacent to dendrites and their location was mainly extracytoplasmic; with the electron microscope, however, some DMS were also found in glial processes. Rounded osmiophilic bodies, 0.1-0.8 microns in diameter, were noticed in mitochondria of both neurons and glial cells; however, whether they were special forms of DMS or different inclusions was not assessed. Both intra- and extracytoplasmic DMS were similar in ultrastructure, appearing as single membrane-bound spherical or pear-shaped bodies containing a cottony or finely granular matrix. Additionally, both perikaryon and processes of large motoneurons were found to contain pigment granules identified as lipofuscin, which seemed to increase in number and to spread centrifugally in the processes in correlation with duration of the intoxication and size of axonal swellings induced by IDPN.(ABSTRACT TRUNCATED AT 250 WORDS)     

THE LOCALIZATION OF ACID PHOSPHATASE IN RAT LIVER CELLS AS REVEALED BY COMBINED CYTOCHEMICAL STAINING AND ELECTRON MICROSCOPY

Discrete localization of stain in pericanalicular granules was found in 10 µ frozen sections of formol-phosphate-sucrose-fixed liver stained by the Gomori acid phosphatase technique and examined in the light microscope. The staining patterns, before and after treatment with Triton X-100 and lecithinase, were identical with those previously reported for formol-calcium-fixed material treated in the same way, and it can be assumed that the stained granules are identical with "lysosomes." Examination in the light microscope of the staining patterns and lead penetration in fixed blocks and slices of various dimensions showed nuclear staining and other artefacts to be present, produced by the different rates of penetration of the various components of the staining medium into the tissue. A uniform pericanalicular staining pattern could be obtained, however, with slices not more than 50 µ thick, into which the staining medium could penetrate rapidly from both faces. The staining pattern produced in 50 µ slices was the same both at pH 5.0 and pH 6.2, and was not altered by subsequent embedding of the stained material in butyl methacrylate. Electron microscopy showed the fine structure of fixed 50 µ frozen slices to be well preserved, but it deteriorated badly when they were incubated in the normal Gomori medium at pH 5.0 before postfixing in osmium tetroxide. After incubation in the Gomori medium at pH 6.2, the detailed morphology was substantially maintained. In both cases lead phosphate, the reaction product, was found in the pericanalicular regions of the cell, but only in the vacuolated dense bodies and never in the microbodies. Not every vacuolated dense body contained lead, and stained and unstained bodies were sometimes seen adjacent to each other. This heterogeneous distribution of stain within a morphologically homogeneous group of particles is consistent with de Duve's suggestion (9) that there is a heterogeneous distribution of enzymes within the lysosome population. It is concluded from these investigations that the vacuolated dense bodies seen in the electron microscope are the morphological counterparts of the "lysosomes" defined biochemically by de Duve.     

Vitamin E attenuates liver injury induced by exposure to lead, mercury, cadmium and copper in albino mice

Water pollution is the contamination of water resources by harmful wastes or toxins. Both community and private sources of drinking water are susceptible to a myriad of chemical contaminants. Heavy metals pollution of surface water can create health risks. The present study was aimed to investigate the effect of vitamin E supplementation on male mice exposed to a mixture of some heavy metals (lead, mercury, cadmium and copper) in their drinking water for seven weeks. Significant increases of blood alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT) were detected in heavy metals-treated mice. Histopathologically, the liver sections from heavy metals-treated mice showed severe changes including disarrangement of hepatic strands, rupture in hepatocytes, advanced hepatocellular necrosis, dilation and congestion of blood vessels with hemorrhage, dense lymphocytic infiltration round the central vein and dark stained hepatocytic nuclei indicating cell pycnosis. Administration of vitamin E at a dose of 50 IU/kg body weight, five times weekly improved the observed biochemical and histopathological changes induced by these heavy metals intoxication. Hence, the results of this study suggest that vitamin E protects against these heavy metals-induced liver injury and the attenuating effect of vitamin E may be due to its antioxidant activity.     

Methamphetamine produces neuronal inclusions in the nigrostriatal system and in PC12 cells

Mice treated with the psychostimulant methamphetamine (MA) showed the appearance of intracellular inclusions in the nucleus of medium sized striatal neurones and cytoplasm of neurones of the substantia nigra pars compacta but not in the frontal cortex. All inclusions contained ubiquitin, the ubiquitin activating enzyme (E1), the ubiquitin protein ligase (E3-like, parkin), low and high molecular weight heat shock proteins (HSP 40 and HSP 70). Inclusions found in nigral neurones stained for alpha-synuclein, a proteic hallmark of Lewy bodies that are frequently observed in Parkinson's disease and other degenerative disorders. However, differing from classic Lewy bodies, MA-induced neuronal inclusions appeared as multilamellar bodies resembling autophagic granules. Methamphetamine reproduced this effect in cultured PC12 cells, which offered the advantage of a simple cellular model for the study of the molecular determinants of neuronal inclusions. PC12 inclusions, similar to those observed in nigral neurones, were exclusively localized in the cytoplasm and stained for alpha-synuclein. Time-dependent experiments showed that inclusions underwent a progressive fusion of the external membranes and developed an electrodense core. Inhibition of dopamine synthesis by alpha-methyl-p-tyrosine (alphaMpT), or administering the antioxidant S-apomorphine largely attenuated the formation of inclusions in PC12 cells exposed to MA. Inclusions were again observed when alphaMpT-treated cells were loaded with l-DOPA, which restored intracellular dopamine levels.     

Morphological alterations in mammalian neurons in vitro in response to hypertonic solutions

Summary Neurons in cultures of central nervous tissue exhibited marked structural changes when exposed to hypertonic solutions. Cellular reactions were described in living neurons as well as after fixation and staining in preparations observed with both the light and electron microscope. The structures involved in these changes were mainly the nucleolus, the nucleus and the Nissl substance.Nucleolus In living neurons, observed with phase contrast optics, the nucleolus became invisible in hypertonic medium. This change occurred within a few seconds, and it was reversible when the cells were brought back to isotonic solutions. Fixation of the cells while exposed to hypertonic solution caused the nucleolus to reappear as a granular body. In stained preparations it appeared as a more irregular body in contrast to the smoothly outlined nucleolus in normal cells. In electron microscopic preparations of neurons which were fixed while exposed to hypertonic solutions the nucleolus was visible only as nucleolar shadow, overlaid by a few small irregular bodies of higher electron density than other nuclear contents.Nucleus The nuclear membrane of living neurons exposed to hypertonic media lost much of its sharp definition and became rather hazy in outline. The nuclear diameter increased about 10% in hypertonic medium, and the nuclear space became somewhat denser when observed with the phase contrast microscope. In Nissl stained preparations the nuclear space was filled with many small granular or rod-shaped bodies in contrast to the clear vesicular appearance of the nuclei of untreated cells. In electron microscopic preparations the nuclear space exhibited a spotty appearance due to the presence of electron dense and light areas.Nissl Substance In living neurons immersed in hypertonic solutions the Nissl substance showed a slight increase in phase density, especially after repeated changes between hypertonic and isotonic solutions. Sometimes a distinct striation in the Nissl substance appeared. In Nissl stained preparations there was no marked change observed in comparison with normal cells. However, in the electron microscope, the Nissl substance of hypertonically treated cells exhibited a marked structural change. The membrane-bound spaces of the endoplasmic reticulum assumed a rather precise orientation parallel to the cell membrane so that in extreme cases a concentric arrangement of endoplasmic cisternae was observed. The normal arrangement of ribosomal granules in rosettes and clusters became disturbed and the granules were more uniformly distributed.The cells as whole units showed a distinct shrinkage in hypertonic solution which may account for the more crowded appearance of various organelles such as mitochondria and Golgi complexes. There was also a marked increase in agranular reticulum profiles and small membrane bound vesicles in treated cells. Vacuoles appeared frequently in the cytoplasm of treated cells; they disappeared upon re-immersion in isotonic medium.This investigation was supported by USPHS Grants NB 03114-04, NB 00690-11 and 5 T 1 GM 495 from the National Institutes of Health, Bethesda, Maryland.Acknowledgement. Mrs. Eleanor W. Morris and Mr. Edwin E. Pitsinger, Jr. gave indispensible aid with the management of the cultures and with photographic procedures.     

Toxoplasma gondii: cross-immunity against the enteric cycle

Eight of nine cats inoculated with strain ME-49 and challenged with three different strains of Toxoplasma were immune to oocyst shedding, as ascertained with bioassays of their feces. In a second experiment, only toxoplasma asexual stages were seen in H&E stained gut sections of cats treated with suppressive doses of sulfamerazine and pyrimethamine starting 2 days after oral inoculation with cysts of the strain ME-49 and killed 6 days later. In a third experiment, four cats were similarly inoculated and treated for 20 days. Six weeks later, the cats received an oral homologous challenge with cysts, and none shed toxoplasma oocysts. An acceptable level of cross-protection was achieved with strain ME-49, and therefore, it can be used as a candidate strain from which antigens could be tested for enteric protection.     

Fluorescence of fungi in superficial and deep fungal infections

Background  

Fluorescence of many fungi is noted when H&E stained sections are examined under a fluorescent microscope. In theory, this phenomenon could aid in the diagnosis of cutaneous and disseminated fungal infections without the delay associated with special stains. Seventy-six cases of superficial and deep fungal infections and 3 cases of protothecosis were studied to determine the clinical usefulness of this technique.     

Overproduced <Emphasis Type="Italic">Brucella abortus</Emphasis> PdhS-mCherry forms soluble aggregates in <Emphasis Type="Italic">Escherichia coli</Emphasis>, partially associating with mobile foci of IbpA-YFP

Background  

When heterologous recombinant proteins are produced in Escherichia coli, they often precipitate to form insoluble aggregates of unfolded polypeptides called inclusion bodies. These structures are associated with chaperones like IbpA. However, there are reported cases of "non-classical" inclusion bodies in which proteins are soluble, folded and active.     

Towards revealing the structure of bacterial inclusion bodies

Protein aggregation is a widely observed phenomenon in human diseases, biopharmaceutical production, and biological research. Protein aggregates are generally classified as highly ordered, such as amyloid fibrils, or amorphous, such as bacterial inclusion bodies. Amyloid fibrils are elongated filaments with diameters of 6–12 nm, they are comprised of residue-specific cross-β structure, and display characteristic properties, such as binding with amyloid-specific dyes. Amyloid fibrils are associated with dozens of human pathological conditions, including Alzheimer disease and prion diseases. Distinguished from amyloid fibrils, bacterial inclusion bodies display apparent amorphous morphology. Inclusion bodies are formed during high-level recombinant protein production, and formation of inclusion bodies is a major concern in biotechnology. Despite of the distinctive morphological difference, bacterial inclusion bodies have been found to have some amyloid-like properties, suggesting that they might contain structures similar to amyloid-like fibrils. Recent structural data further support this hypothesis, and this review summarizes the latest progress towards revealing the structural details of bacterial inclusion bodies.Key words: bacterial, inclusion bodies, amyloid fibrils, protein aggregation, amyloid-like, nuclear magnetic resonance, electron microscope, X-ray diffraction, hydrogen/deuterium exchange, cross-β     

Nuclear death in living Tetrahymena: the case of the haploid nuclei

During sexual conjugation in Tetrahymena the micronucleus divides meiotically, producing four haploid nuclei. While one of these nuclei divides mitotically to yield two genetically identical gametic pronuclei, a stationary pronucleus and a migratory pronucleus, the remaining three haploid nuclei degenerate and disappear. Typically, they migrate to the posterior end of the cell where they remain as residual bodies until they disappear. In the present study we asked whether degenerating haploid nuclei share any properties with apoptotic nuclei. Specifically, we wondered whether they would be stained by "apofluor", a combination of vital fluorescent indicators that differentially stains apoptotic nuclei in living cells. "Apofluor" includes acridine orange, which becomes trapped in acidic compartments and stains lysosomal bodies a brilliant orange-red, and Hoechst 33342, which binds to DNA and stains nuclei bright blue. With this dye combination, while ordinary nuclei stain blue, the apoptotic macronucleus stains first blue-green, then yellow, and finally orange. The progression in color is presumed to be due to the accumulation of protons in the apoptotic nucleus compartment. We found that three of the four post-meiotic haploid nuclei, those that are eliminated, were stained differentially green, then yellow, and then come to be indistinguishable from the orange lysosomal bodies. Differential staining can occur even while the nuclei are located at the anterior ends of the cells, and before the "viable" nucleus divides to form pronuclei. These results indicate that haploid nuclei in the process of degradation are differentially stained in living cells by "apofluor", and that the differential staining occurs early in the elimination process. Further, since the degenerating haploid nuclei are stained by "apofluor" it is likely that they are degraded by a mechanism similar to the elimination of the apoptotic macronucleus.     

Immunodetection of occult eosinophils in lung tissue biopsies may help predict survival in acute lung injury

Background

Acute lung injury (ALI) is a serious respiratory disorder for which therapy is primarily supportive once infection is excluded. Surgical lung biopsy may rule out other diagnoses, but has not been generally useful for therapy decisions or prognosis in this setting. Importantly, tissue and peripheral blood eosinophilia, the hallmarks of steroid-responsive acute eosinophilic pneumonia, are not commonly linked with ALI. We hypothesized that occult eosinophilic pneumonia may explain better outcomes for some patients with ALI.

Methods

Immunohistochemistry using a novel monoclonal antibody recognizing eosinophil peroxidase (EPX-mAb) was used to assess intrapulmonary eosinophil accumulation/degranulation. Lung biopsies from ALI patients (n = 20) were identified following review of a pathology database; 45% of which (i.e., 9/20) displayed classical diffuse alveolar damage (ALI-DAD). Controls were obtained from uninvolved tissue in patients undergoing lobectomy for lung cancer (n = 10). Serial biopsy sections were stained with hematoxylin and eosin (H&E) and subjected to EPX-mAb immunohistochemistry.

Results

EPX-mAb immunohistochemistry provided a >40-fold increased sensitivity to detect eosinophils in the lung relative to H&E stained sections. This increased sensitivity led to the identification of higher numbers of eosinophils in ALI patients compared with controls; differences using H&E staining alone were not significant. Clinical assessments showed that lung infiltrating eosinophil numbers were higher in ALI patients that survived hospitalization compared with non-survivors. A similar conclusion was reached quantifying eosinophil degranulation in each biopsy.

Conclusion

The enhanced sensitivity of EPX-mAb immunohistochemistry uniquely identified eosinophil accumulation/degranulation in patients with ALI relative to controls. More importantly, this method was a prognostic indicator of patient survival. These observations suggest that EPX-mAb immunohistochemistry may represent a diagnostic biomarker identifying a subset of ALI patients with improved clinical outcomes.     

Real time detection and quantification of inclusion bodies expressed in Escherichia coli by impedance measurements

Escherichia coli expressing human growth hormone as inclusion bodies was cultured in a fermenter. Real time, non-invasive detection and quantification of inclusion body protein expressed in E. coli was performed by impedance measurements at 50 MHz and 180 MHz. At 50 MHz rotation of dipoles of the protein and their proton fluctuation, i.e., -dispersion of protein aggregates formed inside the cell as a result of expressed protein, results in an additional decrease in impedance. At 180 MHz the impedance remained at a plateau. In a high cell density E. coli culture, after induction with IPTG, when the cell mass remained unchanged, an increase in the magnitude of -dispersion was observed at 50 MHz. This was due to the formation and subsequent increase in the concentration of r-human growth hormone which aggregate as inclusion bodies. The estimation of inclusion bodies by taking the ratio of impedance at 180 MHz and at 50 Mhz matched with the amount of protein estimated after extraction and purification (coefficient of correlation was 0.92). This is the first report of real time detection and monitoring of recombinant protein expressed as inclusion bodies by impedance measurements.     

Application of a two-phenotype color-shift model with heterogeneous growth to a rat hepatocarcinogenesis experiment

The color-shift model (CSM) was introduced by Kopp-Schneider et al. [1] to describe formation and progression of foci of altered hepatocytes (FAH). It incorporates the field-effect hypothesis which postulates that entire colonies of altered hepatocytes simultaneously alter their phenotype. In the original CSM, FAH grow with deterministic growth rate and change their phenotype after an exponentially distributed waiting time. A modification of the original color-shift model (CSMbeta) is presented here in which the growth rate varies from focus to focus according to a beta distribution. The concept of an exponentially distributed waiting time to phenotype change is modified to the concept of a random radius at which phenotype changes and this radius is modelled as beta distributed. The original and the modified CSM are applied to data from an initiation-promotion rat hepatocarcinogenesis experiment with diethylnitrosomorpholine (DEN) and N-nitrosomorpholine (NNM), in which two phenotypes of FAH were observed in hematoxilin/eosin (H&E) stained liver sections. The Cramer-von-Mises Distance is used as a measure for the discrepancy between empirical and theoretical size distributions. Comparisons of model fit show that considerable improvement is obtained for CSMbeta compared to the original CSM.     

Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F(1)F(o) ATP synthase

Recombinant membrane proteins in Escherichia coli are either expressed at relatively low level in the cytoplasmic membrane or they accumulate as inclusion bodies. Here, we report that the abundant over-production of subunit b of E. coli F(1)F(o) ATP synthase in the mutant host strains E. coli C41(DE3) and C43(DE3) is accompanied by the proliferation of intracellular membranes without formation of inclusion bodies. Maximal levels of proliferation of intracellular membranes were observed in C43(DE3) cells over-producing subunit b. The new proliferated membranes contained all the over-expressed protein and could be recovered by a single centrifugation step. Recombinant subunit b represented up to 80% of the protein content of the membranes. The lipid:protein ratios and phospholipid compositions of the intracellular membranes differ from those of bacterial cytoplasmic membranes, and they are particularly rich in cardiolipin.     

Structure of maize protein bodies and immunocytochemical localization of zeins

Summary Zeins, the seed storage proteins of maize (Zea mays L.), are synthesized by membrane-bound polyribosomes and transported into the lumen of the endoplasmic reticulum in developing endosperm, where they assemble into protein bodies. To better understand the organization of protein bodies and the mechanism by which zeins are assembled, we have used immunolocalization to study their distribution within isolated protein bodies. In sections stained with uranyl acetate and lead citrate, the protein body matrix consists of light- and dark-staining regions with the darker stain predominating at the periphery and the lighter stain in the central region. Immunogold staining of the storage proteins in isolated protein bodies reveals a distinct segregation with -zein localized in the light-staining region and - and -zein localized in the dark-staining regions. However, the relative amounts and distribution of these proteins varies substantially among different protein bodies. These results indicate a more complex internal organization than has been previously observed, and suggest that spatial and/or temporal differences in zein synthesis account for this complexity.Abbreviations BSA bovine serum albumin - IgG immunoglobulin G - PB phosphate buffer - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TTBS Tween-20/tris-buffered saline - TBS-T Tris-buffered saline/Tween-20 - TBS-T-B Tris-buffered saline/Tween-20/bovine serum albumin