Postadhesive Technique for Archival Paraffin Sections

We present a postadhesive protocol for adhering paraffin sections of archival material to microscope slides. Appropriately posttreated sections, subsequently processed for immunohistochemistry, remained attached to the slides and were well preserved with no signs of artifacts, such as scratching and shrinkage. The immunohistochemical staining was intense and antigen-specific without nonspecific background. Specific staining intensity was equal to that produced in untreated control sections; however, the latter became partially or fully detached from the slides. The postadhesion protocol may be used with modern techniques and is recommended for reclaiming use of otherwise unsuitable paraffin sections of archival material.     

A new method for transfer of polyethylene glycol-embedded tissue sections to silanated slides for immunocytochemistry

Polyethylene glycol (PEG) is an excellent embedding medium for immunohistochemical studies. It provides structural preservation superior to frozen sections and increased sensitivity of antigen detection compared with paraffin sections. One limitation of PEG embedment is that PEG sections are difficult to handle and adhere poorly to glass slides. Here we present a simple and effective method for embedding tissues in PEG and transferring the resultant sections onto silanated glass slides. In addition, a method for silver enhanced colloidal gold immunostaining was combined with common dye staining to demonstrate the excellent structure preservation and sensitive antigen detection. Bovine chorionic membrane was fixed with Bouin's fixative, embedded in polyethylene glycol (PEG) 1500, cut into 5-microns sections, flattened over agarose blocks (10 x 10 x 2 mm3), and blotted onto Digene silanated slides. Slides were then washed in PBS, which removed the PEG and agarose blocks. Tissue sections were immunocytochemically stained with dilute antiserum raised in a rabbit against purified bovine placental retinol binding protein (bpRBP). Sections were washed and incubated with 1-nm colloidal gold-labeled goat anti-rabbit IgG. The immunogold particles were enhanced by silver staining (IGSS). Specimens were observed and photographed with an Olympus epipolarization microscope. The new method offered excellent morphological preservation of cell structure and the epipolarization microscopy provided high sensitivity for detection of specific immunogold-silver particles.     

Terminal transferase-dependent PCR: a versatile and sensitive method for in vivo footprinting and detection of DNA adducts.

We report here a new, sensitive and versatile genomic sequencing method, which can be used for in vivo footprinting and studies of DNA adducts. Starting with mammalian genomic DNA, single-stranded products are made by repeated primer extension; these products are subjected to homopolymeric ribonucleotide tailing at the 3' termini with terminal deoxynucleotidyl transferase and then ligated to a double-stranded linker having a complementary 3' overhang, and used for PCR. This terminal transferase-dependent PCR (TDPCR) method can generate band signals many-fold stronger than conventional ligation-mediated PCR (LMPCR). A UV photofootprint in the mouse Xist gene promoter can be easily detected using TDPCR. No special enzymes or chemical reagents are needed to convert DNA adducts into strand breaks. Any lesion that blocks primer extension should be detectable.     

Modified Papanicolaou staining protocol with minimum alcohol use: a cost‐cutting measure for resource‐limited settings

S. Gupta, K. L. Chachra, P. Bhadola and P. Sodhani
Modified Papanicolaou staining protocol with minimum alcohol use: a cost‐cutting measure for resource‐limited settings Objective: To devise a simple, cost‐effective protocol for Papanicolaou (Pap) staining of cervicovaginal smears. Methods: Five hundred coded paired cervical smears were collected from women as part of routine cervical cancer screening. One set of smears was stained by conventional Pap staining protocol (CP) and the other by a modified protocol (MP) in which alcohol was replaced by 1% acetic acid in all the steps except during fixation and prior to mounting; in addition, one alcohol‐based counterstain, OG, was omitted. The smears were examined blindly by the pathologists and then decoded. Each pair of smears was compared and the two protocols were analysed for staining quality and diagnoses by McNemar and chi‐square tests. Results: The staining quality in the MP was satisfactory. The nuclear and cytoplasmic features were comparable to the CP. Cytoplasmic transparency was maintained in the MP and the differential staining of blue/green and pink was acceptable to the pathologists and technicians. The diagnoses agreed in all cases and there was no compromise in interpreting the smears. With MP it took only 3–4 minutes to stain a batch of 50 slides. in contrast to the 20 minutes taken by CP. The MP used almost one‐seventh of the amount of alcohol compared with CP, which translated into a significant cost reduction per smear. Conclusions: The improvised Pap staining protocol with minimum alcohol use is a simple, cost‐effective and technician‐friendly procedure that can be easily adopted in high‐volume, resource‐limited laboratories for mass cervical cancer screening.     

Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.

We describe a new approach for retrieval of antigens from formalin-fixed, paraffin-embedded tissues and their subsequent staining by immunohistochemical techniques. This method of antigen retrieval is based on microwave heating of tissue sections attached to microscope slides to temperatures up to 100 degrees C in the presence of metal solutions. Among 52 monoclonal and polyclonal antibodies tested by this method, 39 antibodies demonstrated a significant increase in immunostaining, nine antibodies showed no change, and four antibodies showed reduced immunostaining. In particular, excellent immunostaining results were obtained with a monoclonal antibody to vimentin as well as several different keratin antibodies on routine formalin-fixed tissue sections after pre-treatment of the slides with this method. These results showed that after antigen retrieval: (a) enzyme predigestion of tissues could be omitted; (b) incubation times of primary antibodies could be significantly reduced, or dilutions of primary antibodies could be increased; (c) adequate staining could be achieved in long-term formalin-fixed tissues that failed to stain by conventional methods; and (d) certain antibodies which were typically unreactive with formalin-fixed tissues gave excellent staining.     

Long-term storage and regular repeated use of diluted antisera in glass staining jars for increased sensitivity, reproducibility, and convenience of single- and two-color light microscopic immunocytochemistry

A procedure is described for the dilution and storage of antisera in glass staining jars into which whole slides are immersed for incubation during light microscopic neuropeptide immunocytochemistry. Diluted antisera, stored at 4 degrees C and continuously reused, were found to be stable for long periods of time (to date over 3 years), and consistently yielded high quality staining in both single- and two-color immunoperoxidase staining. We found this procedure to be more convenient than conventional incubation procedures, allowing the more rapid processing of large numbers of slides and reducing the loss of slides due to technical errors. The consistency and reproducibility of day to day staining were also improved. The immersion of whole slides into the antisera permitted the use of long incubation times (up to 7 days) without the sections drying out, which in many cases substantially enhanced the sensitivity of the staining obtained. A procedure for two-color immunoperoxidase staining is described using diaminobenzidine for a brown color and alpha-naphthol/pyronin for a red/purple color. We found the alpha-naphthol/pyronin reaction superior to the more commonly used 4-chlornaphthol reaction as a second color. The two-color staining was found useful not only for demonstrating nerve cell bodies stained different colors, but also for staining nerve terminals one color that are around and contacting nerve cell bodies stained another color.     

A method for immunohistochemical detection of osteogenic markers in undecalcified bone sections

To evaluate the osteogenic potential of novel implant materials, it is important to examine their effect on osteoblastic differentiation. Characterizing the tissue response at the bone-biomaterial interface in vivo at a molecular level would contribute significantly to enhancing our understanding of tissue integration of endosseous implant materials. We describe here a new technique that overcomes difficulties commonly associated with performing immunohistochemistry on undecalcified sawed sections of bone. Sheep mandible specimens were fixed in an ethanol based fixative to maintain adequate antigenicity of the tissue. As a result, it was possible to omit antigen retrieval at high temperature for recovery of antigenicity, and detachment of sections from the slides was avoided. Following dehydration and infiltration, the specimens were embedded in a resin composed of polymethylmethacrylate and polybutylmethacrylate. Polymerization was achieved by adding benzoylperoxide and N,N-dimethyl-toluidine. This resin was selected because it maintained the antigenicity of the tissue, provided adequate properties for cutting 50 µm thick sections, and it facilitated deacrylizing the sawed sections. Acid-resistant acrylic slides were glued to the blocks using an epoxy resin based two-component adhesive to avoid detachment of the slides during the deacrylation procedure. Samples were stained for alkaline phosphatase, type I collagen, osteonectin, osteopontin, osteocalcin and bone sialoprotein. The EnVision + ™ dextran polymer conjugate two-step visualization system was applied for immunohistochemical detection of these bone matrix proteins. This procedure yielded positive staining for the osteogenic markers in cells and matrix components. The protocol described here facilitates the use of immunohistochemistry on resin embedded sawed sections of bone and provides a convenient and reliable method that can be used routinely for immunohistochemical analysis of hard tissue specimens containing implant materials.     

Protocol for the combination of neurohistological techniques on vibratome obtained sections

Introduction. The histological study of the nervous system requires the use of special techniques. Currently, no methods are available to visualize simultaneously all the cellular constituents of nervous tissue. Objectives. A protocol was adapted for staining nervous tissue by modification of a formerly difficult procedure. Materials and methods. Slices of brain and spinal cord, 4 mm thick, were taken from adult mice, previously fixed by intracardiac perfusion with 4% paraformaldehyde. Vibratome sections were obtained with thickness of 15-25 μm. These were mounted on glass slides prepared with gelatin as an adhesive. The preparations were subjected to staining protocol Luxol Fast Blue-PAS-hematoxylin (LPH) combined with silver staining method (LPH-Holmes). Results. LPH technique yielded an excellent differentiation of gray and white matter in all regions of the nervous system. A panoramic view of the gray matter was colored pink in contrast to the myelinated nerve fibers and tracts which were light blue. The combination LPH-Holmes retained the staining characteristics but significantly improved the demarcation of axons and tracts. Conclusions. A protocol was standardized for the LPH and LPH-Holmes nervous tissue stains applied in combination to tissue slices obtained with a vibratome. The method was shorter, less wasteful and less expensive than the original and also better preserved the integrity of nervous tissue.     

An Activated GOPS‐poly‐L‐Lysine‐ Coated Glass Surface for the Immobilization of 60mer Oligonucleotides

To explore a method for enhancing the immobilization and hybridization efficiency of oligonucleotides on DNA microarrays, conventional protocols of poly‐L‐lysine coating were modified by means of surface chemistry, namely, the slides were prepared by the covalently coupling of poly‐L‐lysine to a glycidoxy‐modified glass surface. The modified slides were then used to print microarrays for the detection of the SARS coronavirus by means of 60mer oligonucleotide probes. The characteristics of the modified slides concerning immobilization efficiency, hybridization dynamics, and probe stripping cycles were determined. The improved surface exhibited high immobilization efficiency, a good quality uniformity, and satisfactory hybridization dynamics. The spotting concentration of 10 μmol/L can meet the requirements of detection; the spots were approximately 170 nm in diameter; the mean fluorescence intensity of the SARS spots were between 3.2 × 10⁴ and 5.0 × 10⁴ after hybridization. Furthermore, the microarrays prepared by this method demonstrated more resistance to consecutive probe stripping cycles. The activated GOPS‐PLL slide could undergo hybridization stripping cycles for at least three cycles, and the highest loss in fluorescence intensity was found to be only 11.9 % after the third hybridization. The modified slides using the above‐mentioned method were superior to those slides treated with conventional approaches, which theoretically agrees with the fact that modification by surface chemistry attaches the DNA covalently firmly to the slides. This protocol may have great promise in the future for application in large‐scale manufacture.     

Immuno-spin trapping analyses of DNA radicals

Immuno-spin trapping is a highly sensitive method for detecting DNA radicals in biological systems. This technique involves three main steps: (i) in situ and real-time trapping of DNA radicals with the nitrone spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), thus forming DMPO-DNA nitrone adducts (referred to here as nitrone adducts); (ii) purification of nitrone adducts; and (iii) analysis of nitrone adducts by heterogeneous immunoassays using Abs against DMPO. In experiments, DMPO is added prior to the formation of free radicals. It diffuses easily through all cell compartments and is present when DNA free radicals are formed as a result of oxidative damage. Due to its low toxicity, DMPO can be used in cells at high enough concentrations to out-compete the normal reactions of DNA radicals, thus ensuring a high yield of DNA nitrone adducts. Because both protein and DNA nitrone adducts are formed, it is important that the DNA be pure in order to avoid misinterpretations. Depending on the model under study, this protocol can be completed in as few as 6 h.     

Rapid staining of proteins on polyacrylamide gels and nitrocellulose membranes using a mixture of fluorescent dyes

The present work describes a novel, fluorescence-based method for staining proteins on SDS-PAGE and membrane(s). In this method, proteins are stained using a mixed-dye (sulfo-rhodamine B and 1-anilino-8-naphthalene sulfonic acid (NH(4)(+))) solution. The mixed-dye staining protocol can detect proteins up to a concentration of 15 ng. This method is generally applicable to all proteins and is more sensitive than the conventional Coomassie blue method. The staining method is rapid and efficient. Staining-destaining of proteins using the mixed-dye protocol takes less than half an hour. Another interesting feature of the staining protocol described here is the applicability to the staining of proteins on nitrocellulose membranes.     

Hot Melt “Corner Point Method” for Attaching Large Plastic Sections to Glass Slides

We describe a fast method for firm attachment of large plastic sections to glass slides with EVA-copolymers, commonly known as hot melt sticks. Solid hot melt sticks dissolve slowly in xylene to form an adhesive gel within 6 hours. Small drops of hot melt gel are applied to the corners of the sections and surrounding slide surface at ambient or elevated temperatures. The gel sticks to both the plastic and the glass slides. The hot melt “corner point method” prevented detachment of sections in staining procedures. As an additional technique, we suggest the use of hot melt adhesive for attaching plastic specimen blocks to wooden blocks or metallic specimen holders.     

Filtration-based staining of proteins on membranes

A filtration-based staining method has been developed for sensitive estimation of proteins in a batch of samples. It is based on focused absorption of an applied protein standard (or sample) and dye solution on nitrocellulose membrane through an aqueous network of capillary channels formed between the membrane and a wetted filter paper. The method does not require any equipment for creating vacuum. Compared with the conventional pouring methods, the new approach reduces the consumption of staining solution and lowers the staining and destaining times (from 1.5 h to ∼ 10 min) without compromising the sensitivity.     

Immunohistochemistry: paraffin sections using the Vectastain ABC kit from vector labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.     

A simple method for enhancing hybridization efficiency in chromosome and array comparative genomic hybridization

Abstract

The accuracy of comparative genomic hybridization (CGH) analysis is affected by hybridization efficiency. We describe here a simple method for enhancing hybridization efficiency. The hybridization procedure is essentially the same as that of conventional methods. Hybridization solution containing denatured DNA probe mixture was applied to a metaphase chromosome slide or DNA chip slide and covered with a coverslip. In the new method, however, the slide was inverted by turning the coverslip downward prior to hybridization. We termed this method the inverted slide method. To estimate the efficiency of the new method, metaphase chromosome slides and DNA chip slides were treated by both the conventional and inverted slide methods and incubated in a moist chamber at 37°C for 12, 24, 48, and 72 h. Hybridization signals were approximately 1.5 to 2 times brighter on the slides using the inverted slide method than those using the conventional method after 48 and 72 h of incubation. Furthermore, topographical differences in fluorescence intensity were smaller in slides using the inverted-slide method than in those prepared by the conventional method. The inverted slide method is methodologically very simple and improves the resolution of CGH.     

Improvement of ciliate identification and quantification: a new protocol for fluorescence in situ hybridization (FISH) in combination with silver stain techniques

A new protocol for taxon specific probe based fluorescent in situ hybridization was developed for the identification and quantification of ciliates in microbial communities. Various fixatives and experimental parameters were evaluated and optimized with respect to cell permeability and morphological preservation. Optimum results were adaption by obatined of a modified fixation method using Bouin's solution. Furthermore, conventional staining procedures such as different Protargol stain techniques and a silver nitrate impregnation method were modified and can now be applied in combination with fluorescence in situ hybridization. The new protocol allows a rapid and reliable identification as well as quantification of ciliates based upon classical morphological aspects and rRNA based phylogenetic relationships performed in one experiment. Furthermore, a set of specific probes targeting different regions of the 18S rRNA was designed for Glaucoma scintillans Ehrenberg, 1830 and tested by applying this new approach of combining in situ cell hybridization with conventional staining techniques.     

External quality control for immunocytochemistry on cytology samples: a review of UK NEQAS ICC (cytology module) results

I. S. Kirbis, P. Maxwell, M. S. Fle?ar, K. Miller and M. Ibrahim External quality control for immunocytochemistry on cytology samples: a review of UK NEQAS ICC (cytology module) results Objective: To date, external quality control for immunocytochemistry on cytology samples is provided only by the United Kingdom national external quality assessment service for immunocytochemistry and in situ hybridisation (UK NEQAS ICC & ISH). For the purpose of this study a retrospective analysis of a comprehensive collection of quality‐related data regarding immunocytochemistry on cytology samples collected through this service was analysed. Methods: The quality of immunocytochemical reactions, using on‐line collected data, was analysed for the last 23 UK NEQAS ICC cytology module external quality assessments carried out on cytology samples completed in the period from 2004 to 2010. Results: Our study showed that the majority of participants in the cytology module (66%) sent formalin‐fixed paraffin‐embedded (FFPE) tissue sections for assessment as in‐house control slides and only 34% sent cytology slides of various types. The highest UK NEQAS ICC score for the quality of immunocytochemical staining among in‐house control slides was achieved on cell block sections, followed by cytospins, FFPE tissue sections, liquid‐based cytology slides and smears. With regard to fixation, acetone‐fixed slides achieved significantly lower scores than other reported fixatives. The strength of agreement in perception of immunocytochemical staining quality was good between in‐house assessors (Kappa = 0.64) but only fair between in‐house and UK NEQAS ICC assessors (Kappa = 0.22). Conclusions: Good quality of immunocytochemical staining can be achieved on cytology slides prepared and fixed in different ways as well as on cell blocks. Unified criteria for high‐quality immunocytochemical staining and proper internal and external quality assurance could facilitate further improvement and standardization of immunocytochemistry on cytology samples.     

Method for Staining and Washing Thin Sections on Support Films

A procedure is described for staining large numbers of thin sections on support films for use with one-hole grids. The film is picked up, carried and protected using easily made plastic blocks. Loop-tipped forceps are then used to transfer tissue ribbons from the knife boat to the support film. A large number of tissue sections can then be stained and washed simultaneously in a modified Pyrex dish without damaging the film. After staining, the slot in the one-hole grid is centered over the tissue ribbon, and the grid is attached to the film. The method is suitable for serial reconstruction and the unobstructed viewing of large thin sections in the TEM.     

The 32P-postlabeling assay for DNA adducts

32P-postlabeling analysis is an ultrasensitive method for the detection and quantitation of carcinogen-DNA adducts. It consists of four principal steps: (i) enzymatic digestion of DNA to nucleoside 3'-monophosphates; (ii) enrichment of the adduct fraction of the DNA digest; (iii) 5'-labeling of the adducts by transfer of 32P-orthophosphate from [gamma-32P]ATP mediated by polynucleotide kinase (PNK); (iv) chromatographic or electrophoretic separation of the labeled adducts or modified nucleotides and quantitation by measurement of their radioactive decay. The assay requires only microgram quantities of DNA and is capable of detecting adducts at frequencies as low as 1 in 10(10) nt, making it applicable to the detection of events resulting from environmental exposures, or experiments using physiological concentrations of agents. It has a wide range of applications in human, animal and in vitro studies, and can be used for a wide variety of classes of compound and for the detection of adducts formed by complex mixtures. This protocol can be completed in 3 d.