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Index
Accuracy, of image analysis, 186-187 Acetic anhydride, for pretreatment, to
reduce background hybridization, 122
Acrolein, for tissue preservation, 15 Acrylic resins, for embedding tissue
for immunocytochemical studies, 16-17 at low temperatures, 21
Adhesion molecules, 231-233 Adhesives, for coating slides, 31-32
in antigen retrieval, 61 for cell smears, 36-37
Adrenomedullin detection of
in the islets of Langerhans, 167-168 in rat brain, 161-163
mRNA for, localization in human lung, 158-161
Advisory Committee on Dangerous Pathogens (ACDP), classification system of, 221
Affinity labeling applications in biomedical sciences,
223-253 defined, 2, 75 list of systems, with references, 225 procedures for identifying cell cycle
status, 229 Agar 100, for electron microscopy, with
osmicated samples, 43 Agar diffusion technique, for immuno
negative staining, 41 Agarose gel electrophoresis, for checking
the size of cRNA preparations, 117
Alcohols, disposal regulations for, 215 Aldehydes, for tissue preservation, 12-13.
See also Glutaraldehyde Alkaline hydrolysis, for preparing probe
RNA fragments, 117 Allergy
to animals, sample contamination with allergens, 208
to laboratory animals, 220 Aminopropyl triethoxy silane (APES), as
an adhesive for coating slides, 31-32,61
Ammonium molybdate, for immunonegative staining, 39-40
Amplification, of target nucleic acids, 12lf. See also Gene amplification; Polymerase chain reaction
Amyloid plaques ~, in Alzheimer disease, immunocyto
chemical identification of, 246 enhancement of images of, 181-183
Analysis of variance, 190-191 Aneuploidy studies, with fluorescence in
situ hybridization, 150-151 Annexins, monoClonal antibodies to, for
quantifying apoptotic cells, 230 Antibodies
defined, 74-75 secondary, amplification of signals
using, 124 techniques for using
with markers, 78-83 without markers, 76-78
Antigen decoration, 78
255
256 Index
Antigenic sites, preservation for immunocytochemistry, 6-29
Antigens defined, 74 exposing for immunolabeling, 30 immobilizing while preserving immu-
noreactivity of, 6-7 localization of, protocols for, 9 retrieval of, 55-72
Apoptosis affinity labeling for studying, 229-230 evaluating role in chronic hepatitis C
injury, 245-246 Apoptotic rheostat, 230 Applications
of affinity labeling in biomedical sciences, 223-253
of fluorescence in situ hybridization technology, 148-151
Araldite, hazards of using, 215 Area measurement, in stained specimens,
177 Artifacts, in nonradioactive in situ hybrid
ization, 125-128 Autoantibodies, circulating, identification
of,247 Autoclave heating, for antigen retrieval,
68 Autometallography (silver enhancement),
87 Avidin-biotin complex (ABC) procedure,
167
Background hybridization, nonspecific, limiting, 125-128
Back-scattered electron detector (BSE detector),52
Bar coding, of chromosomes, 151 Base composition, of probes, and stability
of hybrids, 122 bax, effect on cell survival, 229-230 B cells, tonsil, preserving the antigenicity
of,15 bcl-l, association with mantle celllym
phoma, 235 bcl-2
association with follicular lymphoma, 235
effect on cell survival, 229-230 bcl-6, association with Burkitt's lym
phoma, 235 Bezafibrate, treatment with, and peroxi
somal gene expression, 129 Biological hazards
decontamination of waste, 207 regulations for reducing, 220 from tissues, and fixation procedures,
213 See also Safety precautions
Blocking, to reduce variability in experimental studies, 196
Brain detection of adrenomedullin in, 161-
163 of a piglet with Menangle virus infec
tion, 101 5-Bromodeoxyuridine (BrdU), for cell
synchronization, 140 Buffers, use in preparing specimens for
immunolabeling, 75-76 Burkitt's lymphoma, association with bcl-
6, 235
Cadherins, identifying roles in malignancy, 231
Calcium coordination complexes, masking of antigens by, after formalin fixation, 58-59
Calcium ions, effect on tissue preservation, 13
Calibration, system, prior to analysis, 178-179
Carbodiimide, cross-linking reagent for tissue preparation, 14
Carcinogens, safe procedures for managing in the laboratory, 216-218
Catalase mRNA, specificity of staining for, in liver and kidney, 129
Catenins, localization of, immunocytochemical, 231
CD molecules for affinity labeling, list, 228 CD44, monoclonal antibody labeling of,
in tumor cells, 233 CD45, immunolabeling of, in kidney
graft cryostat sections, 237-240
cDNA analysis of clones, with fluorescence in
situ hybridization, 148-149 probe length determination, 118
Celiac disease, identifying with immunocytochemical serological screening, 247
Cell count, stained cells, 177 Cell cycle, phases of, microscopic markers
for, 228 Celloidin, safety considerations in using,
214 Cells
cytokine-producing, saponin cell permeabilization procedure in identification of, 230-231
effect on survival, of bcl-2, 229-230 preparation of, for fluorescence in situ
hybridization, 139-142 virally infected, immunonegative stain
ing of, 41 Cell smears, preparing, for light micros
copy, 36-37 Cell-surface antigens, human leucocyte,
classification of, 226-227 Cell-surface labeling, 9-10 Charged coupled device (CCD) camera
for fluorescence' in situ hybridization studies, 148
image analysis using, 178-179 Chemical fixation, methods of, 11-16 Chemicals
reactions involving purchased chemicals and solutions, 217
safety considerations in handling, 212-219
Chemicals Hazard Information and Packing for Supply (CHIP) regulations, 202
Chemokines, identifying with monoclonal antibodies, 231
Chromosome paints, 150 Chromosomes
7, changes in acute myeloid leukemia, 151
localization of regions, with fluorescence in situ hybridization analysis, 148-149
Index 257
preparation for fluorescence in situ hybridization, 139-142
preparation of metaphase slides from lymphocyte suspensions, 142
Citrate buffer, heating specimens in, for antigen retrieval, 59
Citrus oils, for dewaxing, 216 Clones, order of, on a chromosome, 149-
150 Clothing, in the laboratory, safety consid
erations, 205 Clusters of differentiation (CDs), for leuco
cytes, 226-227 Coagulants, in fixative solutions, for
preservation of antigenic activity, 14-15
Colcemid, arrest of dividing cells at metaphase, 140
Colloidal gold direct labeling using, 85 for visualization in electron micros
copy, 2 labeling microorganisms with, 80-82 LR, for specimen preparation for immu
nonegative staining, 44 for probes, scanning electron micros
copy, 51-52 Color development, in single step detec
tion of mRNA, 163 Color images, quantitative aspects of, 179 Comparative genomic hybridization
(CGH), 151-152 Compliance, with government safety regu
lations, components of, 202 Composition, of purchased chemicals and
solutions, manufacturer's information about, 216
Computers, 4 Confocal laser scanning microscope, 7
for generating digitized images, in fluorescence in situ hybridization, 148
Connexins, in gap junctions, 236 Consortium to establish a registry for Alz
heimer disease (CERAD), criteria for diagnosis of dementia, 176
Control of contamination by purchased chemi-
258 Index
Control (cont.) cals and solutions, manufacturer, 218 of substances hazardous to health
(COSHH), 200, 202 Controls
for antibody techniques without markers, 76-78
for intensity of labeling, 92-93 in mammalian liver, albumin cRNA,
109-111 Controls (cont.)
negative, mRNA sense probe, 111 for in situ hybridization, 125-128 for in situ polymerase chain reaction,
170--171 Cover slips, artifacts from incorrect use
of, 126 Creutzfeld-Iakob disease (CID), biological
hazard from handling tissues involving,213
cRNA digoxigenin-Iabeled, for detection of
mRNAs, 108-137 Cross-linking reagents, for preserving tis
sue for light microscopy and electron microscopy, 14-15
Cryofixation, defined, 8 Cryoprotectants, for tissue preparation, 20 Cryosections/cryosectioning
frozen substitution, 20--21 preparing, for light microscopy, 34-35
Cryostats, hazards in using and maintaining, 209, 212
Cryoultramicrotomy, 46-48 Cyclin-dependent kinases (CDKs), in the
cell cycle, identifying, 228 Cytokeratin, identifying carcinomas with
monoclonal antibodies to, 234 Cytokines
immunocytochemical characterization of, in dengue fever, 243-244
signaling by, 230--231 Cytological preparations, for light micros
copy, 36-37 Cytomegalovirus (CMV), monitoring
infection with cytospin preparation, 244 using affinity labeling, 240--241
Cytometry, laser scanning, 248 Cytospin preparation of cells, 36
storage of, 38
Data analysis, in immunocytochemistry, qualitative, 175-187
Data extraction, automation of, 185-186 Dehydration
agents for, safe procedures for using in the laboratory, 214
of fixed tissue, in preparation for embedding, 17-18,42
Deproteination, before hybridization, 120--121
Design, experimental, for separating components of variation, 189-192
Dewaxing citrus oils used for, 216 before hybridization, 120
Diaminobenzidine (DAB), carcinogenic properties of, 216
Digitized images, from fluorescence in situ hybridization, 148
Digoxigenin developing, 160--161 as a hapten label, 116-117
Dimethylsuberimidate, for coagulation in tissue preservation, 14
Dimethylsulfoxide, as a cryoprotectant, 20
Direct application, for immunonegative staining, 40
Direct in situ hybridization, 124 Direct in situ polymerase chain reaction,
157-165 Direct labeling, 78-80
with colloidal gold, 85 DNA
damage to, and repair of, 234-237 as a double-stranded probe, 112
relative advantages of, 115 mitochondrial, detecting deletions in,
164 probe, labeling of, 143-147 reverse transcription of, 161
DNA double-strand break repair (DSBR), monoclonal antibody probes for studying, 234
DNA polymerase, Tth, 161
DNAse digestion optimization of, in situ procedures,
168-169 protocol for, 159
Dot blot assay, for measuring digoxigeninlabeled standard RNA, 117-118
Double labeling, for identification of cell phenotype and cytokine receptor, 230-231
Duchenne muscular dystrophy, study of neuromuscular junction pathology in, 246
Duration, of hybridization reactions, and stringency of conditions, 123-124
Ebola virus, immunohistochemistry for studying infection by, 241
Electron microscopy examples of labeling in, 93-97 immunolabeling for use in, 76-87 negative contrast, in antigen decoration,
78 specimen preparation for, 38-51 See also Scanning electron microscopy;
Transmission electron microscopy Embedding media, hazards from using in
the laboratory, 214-215 Embedding tissue, for sectioning, 16-18 Encephalopathy, dengue virus antigen
identificationin,242-243 Environment, laboratory, safety in, 206-
210 Enzyme chromagen systems, 88-89 Enzymes, as markers in immunolabeling,
88-89 Epitopes, resolving the position of, 7-8 Epon, hazards of using, 215 Epoxy resins, for embedding tissue,
effects on epitopes, 16 Epstein-Barr virus (EBV), detecting active
infection by, 245 Erythrocytes, pig, infected and uninfected,
100 Estrogen receptor, localizing, indirect in
situ PCR for, protocol, 165-166 Ethers, precautions in using and storing,
214 Evidence, about safety of purchased
chemicals and solutions, manufacturer's info, 217
Index 259
Exposure limits, of purchased chemicals and solutions, manufacturer's info, 217
Exposure route, and safety of chemicals and solutions, manufacturer's information, 216
False positives, in immunolabeling, preventing, 89
False replication, in experimental design, 191
Families, of cell adhesion molecules, 231 FaslFas-ligand pathway, apoptosis trigger
ing in, 229-230 Filters, effect on images, 180 Fire, involving purchased chemicals and
solutions, 218 Fire regulations, in laboratories, 204-205 First aid, in accidents involving purchased
chemicals and solutions, 217 Fixation
and biological hazard from tissues, 213, 221
crosslinking versus precipitation processes, 118-120
hazards of handling reagents for, 213-214
optimum conditions for, 118-119, 168 purposes of, 75 for single-step detection of mRNA,
161-162 Flow cytometry, for chromosome sorting,
151-152 Fluorescein isothiocyanate (FITC),
annexin V-, for studying nuclear and membrane status, 230
Fluorescence, 2 nonspecific, from trichloroacteic acid
and mercuric chloride fixative, 15 Fluorescence in situ hybridization (FISH),
3, 138-155 Fluorescence microscopy, 147-148 Fluorescent markers, 89-90 Fluorescent probes, signal detection and
visualization, 124 Fluoro-nanogold (FNG) probes, 90 Follicular lymphoma, association with
bc1-2,235 Formaldehyde, disposal procedures, 215
260 Index
Formalin, for fixation of tissues for immunohistochemistry, 15 unmasking of antigens after, 55-56
Forster-type energy transfer effect, in situ 5-nuclease assay, 163-165
Fracture label, methods for preparing, 50-51
Freeze-fracture techniques, for exposing complementary endoplasmic and protoplasmic surfaces, 49-51
Freeze substitution, for preparing samples for immunonegative staining, 42, 48--49
Freezing techniques hazards of, 219-220 for preparing tissue, 18-20
Freezing tissues, for light microscopy, 35-36
and preservation, 38 Fumigation, of cryostats, 212
Gap junctional intercellular communication, role in tumor suppression, 236
Gene amplification methods of, 166-168 in situ, 156-157
Genes, studying expression of, with in situ hybridization, 128-131
Glass dust, cleaning from the laboratory, 208
Glass slides, care in handling, 209 Globin, hybridization for detection of the
gene for, 109 Glutaraldehyde
disposal regulations, 215 tissue preservation using, 12-13
Glycerol, as a cryoprotectant, 20 Gold-fluorochrome antibody complex, 90 Goodpasture syndrome, glomerular base-
ment membrane staining in, 247 Gray level histogram, for selecting objects
of interest, 182 Gray scale, for analyzing images with only
one chromogenic product, 179 Green fluorescent protein (GFP), as a
reporter molecule, 247 Grid cell culture, for examining virally
infected cells, 41
Growth factors, identifying with monoclonal antibodies, 231
Gut-associated lymphoid tissue (GALT), studying leucocyte trafficking in, with monoclonal antibodies, 233
Hapten labels digoxigenin as, 116-117 for DNA probes, 143 for nonradioactive probe labeling, 112
Hazard defined, 200-201 of chemicals and solutions, manufac
turer's information, 217 Hazardous substance
defined, 202 identification of, 203
Health and Safety at Work Act (Britain), 201-202, 204-205, 221
Heart cells from, distribution of fibronectin,
laminin and ribosomes in, 11 muscle of, from a chicken with New
castle disease virus infection, 101 Heat, for antigen retrieval, boiling speci
mens with heavy metal salt solutions, 58-59f. See also Autoclave; Microwave radiation; Pressure cookers; Water bath
Heat shock proteins, expression of, in response to stress, 235-236
Hepatic neoplastic transformation, studying,229
Hepatitis C, chronic, antigen expression in, 245
Herpes simplex virus type I (HSV-l), reactivation and study of, 244-245
Hierarchy, of safety management in the workplace, 204
Hormone receptors, 233-234 Hot plates, maintenance for safety,
209-210 Hot start techniques, for polymerase chain
reaction, 169-170 Howie Report, on handling fresh animal
tissue, 221 Human immunodeficiency virus (HIV),
detection with in situ gene amplification, 156-157
Hybridization conditions for, stability of hybrids, 122-
124 guidelines for developing a protocol for,
109-125 in kidneys, specificity of glyceraldehy
dephosphate dehydrogenase for, 129
Hybrids, types of, and stability, 122 Hypolipidemic drugs, effect on transcrip
tion of genes involved in straightchain fatty acid reactions, 131
Image analysis, 175-199 defined, 177
Immune disorders, pathology of, immunocytochemical studies, 246-247
Immunocytochemistry defined, 1 preservation of tissue for, 6-29 quantitative, 175-199 specimen preparation for, 30-54 typical procedure, safety considerations
step-by-step, 218-219 Immunoelectron microscopy, negative
contrast, 93-95 Immunofluorescence labeling, temporary
nature of, 246-247 Immunoglobulin G superfamily, role in
adherence of leucocytes to blood vessels, 233
Immunoglobulins. See Antibodies Immunogold-silver procedure, to compare
immunophenotypic and morpho-logical leucocyte identification, 237
Immunohistochemistry, preservation of antigenic sites for, 6-29
Immunohistopathology, for lymphoma diagnosis, 234-235
Immunolabeling, 73-107 evolution of, 1-2 postembedding for, 42-46 preembedding for, 41 variables affecting, 3
Immunonegative stain technique, preparing specimens for electron microscopy, 39-41
Index 261
Immunoperoxidase methods, information sheets accompanying use of, 216-218
Immunostaining. See Immunolabeling Imprints, preparing, for light microscopy,
37 In-block labeling, 9-11 Incubation times, 90-91 Indirect labeling, 80, 85-87 Infection, exploitation of cadherins by
pathogens in, 231 Infectious tissue, safe handling of, 220-
221 Information
about leucocyte antigens, online access to, 227
manufacturer's, checklist for determin-ing adequacy of, 216-218
Information-rich experiments, 192-196 In situ 5-nuclease assay, 163-165 In situ hybridization (ISH), 3, 108
indirect, 124 limitations on use in diagnosis, 224
In situ polymerase chain reaction, 224 indirect, 165-166
Inspections, safety, 204 Instruments, safe handling of, in the
cut-up areas, 208 Insulin
hybridization for detection of the gene for, 109
immunolabeling of, for assessing viability of transplanted islets, 239
Integrins, role in inflammation, gutassociated lymphoid tissue (GALT), 233
Interleukins,230-231 Interphase, separation of clones in, identi
fying with fluorescence in situ hybridization, 149-150
Islet of Langerhans, transplantation of identifying viable islets, 241-242 for treating diabetes, 239
Karyotyping, spectral, 151 Kidneys
catalase mRNA in, specificity of staining for, 129
262 Index
Kidneys (cont.) CD45 immunolabeling in graft sections,
and cellular rejection, 237-240 glyceraldehydephoshate dehydrogenase
for hybridization in, 129 Knives, safe handling of, in the laboratory,
208
Label fracture, for visualizing the distribution of externally labeled antigens, 50-51
Laboratory organization of, impact on safety, 205-
206 personnel responsible for safety in,
203-205 Laundry, for laboratory clothing, 207 Lead citrate, safety considerations in use
and disposal of, 215 Legal obligation, to maintain safety, 200 Legislation, health and safety, 201-203 Leidenfrost effect, in freezing tissue, 18-19 Leucocytes
and adhesion molecules, 231-233 characterization of, 224--227
Life cell molecular distillation process, 49 Lighting, safety considerations in design
ing,207 Light microscopy
for immunocytochemistry, 1-2, 87-90 immunolabeling in, 97-\03 specimen preparation for, 31-38
Lineage-specific markers, of leucocytes, 226-227
Lipid metabolism, roles of peroxisome proteins in, 128
Liver, catalase mRNA in, specificity of staining for, 129
London resins, for immunonegative staining,43-44
Lowicryls for preparing specimens for immuno
negative staining, 44--46 toxicity of, 215
LR white resin for immunonegative staining, 43-44 toxicity of, 214
L-selectin, role in leucocyte binding to endothelium, ,232-233
Lung, localization of mRNA for adrenomedullin in, 158-161
Lymphocyte, culturing from blood, protocol, 140-142
Lymphomas, categorizing, 234--235 Lyso1ecithinINonidet permeation proce
dure, for accessing intracellular antigens, 230-231
Management of Health and Safety at Work Regulations, risk assessment duty of, 202
Mantle cell lymphoma, association with bcl-I,235
Melting temperature (T m), calculating, formulas, 122-123
Mercuric salts, avoiding use of in the laboratory, 214
Metaphase slides, preparing from lymphocyte cell suspensions, 142
Methylene bridges, between active sites in aldehyde-fixed tissues, 56
Methyl methacrylate, antigen retrieval from specimens in, 70-71
Microtomes, hazards in using and maintaining, 209
Microtomy, safety considerations in, 212 Microwave pressure cooker, for heating
samples in antigen retrieval, 67-68 Microwave radiation
for fixing tissue, 8, 18 for retrieving antigens, 59-65, 121 safety considerations in using ovens,
210-211 Minimum exposure limits (MEL), for
formaldehyde, venting to maintain, 208
Mismatch repair, monoclonal antibody probes for studying, 234
Mitochondrial DNA, detecting deletions in, 164
Mitogens, for fluorescence in situ hybridization studies, 140
Monoclonal antibodies (MAbs) defined, 74 to epitopes of ribosomal transcription
factor UBF, 228-229 for identifying chemokines, commer
cially available, 231 Monospecific antibodies, defined, 74--75 Morphological preservation, in postem
bedding immunolabeling, 42
mRNA catalase, specificity of staining for, in
liver and kidney, 129 peroxisomal, in situ hybridization pro-
tocol for localizing, 128-129 probes for detecting transcripts, 112 single-step detection of, 161-163 two-step detection of, by direct in situ
reverse transcription-PCR, 158-161
Multiple labeling, 83-87 gold-silver enhancement protocols, 87
Multiplex-fluorescence in situ hybridization (M-FISH), 151
Necroscopy knives, disposable, 208 Necrosis versus apoptosis, methods for
discriminating cell death mechanisms, 230
Neurological studies, with affinity labeling, 246
Neutral buffered formalin (NBF), for fixing tissue for light microscopy, 32
Newcastle disease virus, light microscopy for identifying, 98-99, 101-103
Nick translation, for labeling DNA probes, 143
Nitric oxide synthase, neuronal, at the sites of degeneration of ischemic brain, 246
Nitrocellulose, low-viscosity, for embedding, risks of use, 214
Non-radioactive labeling, with fluorescence in situ hybridization, 139
Nonspecific interactions, in immunolabe1-ing, 92
Nucleic acids, in situ amplification and detection of, 156-174
Nucleolar organizer regions (NORs), 228-229
Nucleotide excision repair (NER), 234-237 Nucleotides, radioactively versus nonradio
actively labeled, 116
Objectivity, of image analysis, versus user interaction, 185-187
Object selection based on gray scale or color, 180-184 based on shape, 184-185
Index 263
Optical density, of reaction products, 176-177
Optical microscopes, range of, 7 Optimization, for in situ gene amplifica
tion, 168-171 Organization, safety concerns in, 203-205 Osmium tetroxide
for cell preservation, 15 hazards to laboratory personnel, 214
disposal regulations, 215 ~-Oxidation pathways
changes in, after injection of fibrate drugs, 131
for oxidation of lipids, 128
p53 tumor-suppressor gene, 234 Parabenzoquinone, for coagulation in
tissue preservation, 14 Paraffin. See Wax entries Particulate markers, 90 Pathways, for apoptosis triggering, 229-
230 PCR. See Polymerase chain reaction Perforin-granzyme B pore-forming
pathway apoptosis triggering in, 229-230 in cellular rejection of transplants,
237-240 Perfusion fixation, 119 Peroxidase, use in immunolabeling, 88-89 Peroxisomal proteins, mRNAs encoding,
localization with in situ hybridization, 128-131
Peroxisome, defined, 128 Peroxisome proliferator-activated receptor
a (PPAR-a), mRNA, 128 Personal protection
against contamination by chemicals and solutions, 218
in the laboratory, 205 See also Safety precautions
Personnel for implementing safety regulations,
203 training of, in safety procedures, 204
Phosphotidylcholine, locating, with label fracture techniques, 50
264 Index
Phosphotungstate, sodium, for immunonegative staining, 39-40
Phytohemagglutinin (PHA), for stimulating cell division, for fluorescence in situ hybridization, 140
Picric acid for coagulation in tissue preservation, 14 safety considerations in handling, 213-
214 Pipettes
disposal of tips, 207-208 safety observation in use of, 205
Plectin, precise location of in the cytoskeletal framework, 8 technique, 10
Polyclonal antibodies, for identifying cytokines, 230-231
POlY-L-lysine, as an adhesive for coating slides, 31-32
Polymerase chain reaction (peR), 3-4 diagnostic use of, 224 protocol, 160 in situ, 224 solution for, optimizing composition,
170 Polymerization, with ultraviolet irradia
tion, for embedding frozen tissue, 21
Polyvalentlpolyclonal antibodies, defined, 75
Polyvinyl pyrrolidone (PVP), to protect specimens from osmotic shock, 44
Postembedding immunogold electron microscopy (POIEM), 95-97
Postembedding labeling, 11 immunolabeling, 42-46
Power, statistical, of a study, 194 Precision, experimental, calculating, 193 Preembedding immunogold electron
microscopy (PRIEM), 95-97 Preembedding immunolabeling, 41 Prehybridization, conditions for, 122 Preimplantation diagnosis, with fluores-
cence in situ hybridization, 151 Prenatal diagnosis, of trisomies, with fluo
rescence in situ hybridization, 151 Pressure cookers
for heating samples for antigen retrieval, 65--68
unmasking antigens in, safety considerations, 211-212
Pretreatment, before hybridization, 120-122, 158-159
Prions, probes reacting with isoforms of, 248
Probes DNA
labeling for fluorescence in situ hybridization, 143-147
size of, and penetration into samples, 117-118
gold silver enhancement of, 97 size of, 80-82
monoclonal antibody, list, 238 preparation and labeling of, 112-118 protein A, for indirect labeling, 85-87 size of, and stability of hybrids, 122
Processing environment, safety considerationsin, 208
Progressive lowering of temperature (PLT) method,17
Propylene oxide disposal regulations, 215 hazards of using, 214
Protease digestion, optimization of, 169 Protein, nonstructural, in bluetongue
virus-infected cells, 95-97 Proteinase K, for digesting proteins
masking mRNA, 120-121, 159, 162
Protein Reviews on the Web (PROW), 227
Proteolytic enzyme digestion for antigen retrieval, 56--58 combining with microwave irradiation,
for unmasking antigens, 62--65 Protocols
for fluorescence in situ hybridization, 139-148
labeling the DNA probe, for fluorescence in situ hybridization, 143-147
P-selectin, role in inflammation reactions, 232
Quality image processing to enhance, 179-180 of science, and statistics, 187-196
Rabies virus antigen, in situ detection of, 242
Radioactive labels artifacts in use of, 126-128 signal detection and visualization, 124
Randomization, in experimental studies, 196
Recombinant antibodies, defined, 75 Refrigerators, spark-proof, for laboratory
use, 213 Region of interest (ROI), 180
in image analysis, 182 Renal transplantation, P-selectin secretion
in, 232-233 Replication, experimental, 192-196 Resin sections
for immunonegative staining, 43 for light microscopy, 33-34
antigen retrieval on, 70-71 safety precautions in preparing, 200-
222 storage of, 38
Resolution, dependence on preservation of tissue properties, 6-7
Reverse chromosome painting, molecular cytogenetics, 151-152
Reverse transcriptase, protocol, 159-160, 162-163
Reverse transcription, of RNAs to cDNAs, 161
Revised European-American classification of lymphoid neoplasms (REAL), 234-235
Ribosomal transcription factor, UBF, monoclonal antibodies to, 228-229
Risk, defined, 200-201 Risk assessment
assigning responsibility for, 202-203 legal requirement for, 202
RNA reverse transcription of, 161 for single-stranded probes, 112
relative advantages of, 115 See also cRNA
Safety precautions in handling fresh unfixed specimens, 55 in handling resins for embedding, 18 in the laboratory, 200-222
Index 265
Sample preparation, for image analysis, 177-178
Sampling, strategy for, 192-196 Saponin cell permeabilization procedure,
in identification of cytokineproducing cells, 230-231
Scalpels, disposable, 208 Scanning electron microscopy (SEM)
methods for, 51-52 types of instruments for, 7 See also Electron microscopy
Science, quality of, and statistics, 187-196
Seating, ergonomic, in the laboratory, 206-207
Sectioning artifacts introduced by problems with,
126 effects of fixation procedure on, 119 embedding tissue for, 16-18 labeling of both faces, 83-84
Selectins, role in attaching leucocytes to blood vessel walls, 231-233
Sequence identity, extent of, and hybrid stability, 122
Sharp tools, contaminated, disposal of, 207
Signal detection, in hybridization studies, 124-125
Signal transduction, intracellular, 227-230 Silver
ammoniacal, care in handling, 215 for staining of nucleolar organizer
regions (AgNORs), 228 Silver enhancement, of small gold probes,
87 Single-copy gene localization
adaptation of tyramide signal amplification to, 125
detection by in situ hybridization, 138-139
Single step detection, of mRNA, 161-163 Slides, preparation of, for light micros
copy, 31-32 Solid-phase immunoelectron microscopy
(SPIEM),76-78 Solutions, for in situ amplification and
detection of nucleic acids, 173-174
266 Index
Solvents containers for, safety considerations, 206 hazards of using, 215-216 inhalation of vapor from, hazard of, 213 reducing the risk of fires involving,
212-213 risks of heating in microwave ovens,
210--211 venting of, from the laboratory, 208
Specific interactions, in immunolabeling, 92
Specificity, of hybridization, 129 Specific operating procedures (SOP), for
explicating hazards and safe working procedures, 204
Specimens fixation of, 2-3 handling, for immunonegative staining,
42 preparing, 118-120
for light microscopy, 32-37 for postembedding immunolabeling,
42 Spills
chemical, procedures for managing, 213 involving purchased chemicals and
solutions, 217 Stains
carcinogenic, handling procedures, 215 reproducible, for image analysis, 178
Standardization, of data interpretation in immunocytochemistry, 175-187
Statistical analysis, in image analysis, 186 Statistics, and good science, 187-196 Steamer heating, for epitope retrieval,
69-70 Steric hindrance, by gold probes, effect of
probe size, 83 Storage
of chemicals, in the laboratory, 212-213 of probes, for fluorescence in situ
hybridization, 143 of specimens, for light microscopy,
37-38 Structural changes, chromosomal, detect
ing with fluorescence in situ hybridization, 150
Sucrose, effect on unmasking antigens in preservation, 15
Surfactants, to facilitate labeling, by exposing tissue detail, 10
Tau immunoreactivity, enhancing in brain tissue, 68
T cells, localization of cytotoxic products of, in transplant rejections, 237-240, 247-248
Temperature of hybridization, and stability of
hybrids, 122 of tissue for substituting organic sol
vents for water, 21 Thresholding, in image analysis, 183 Threshold limit value (TLV), for xylene,
216 Thymidine, for cell synchronization, in
fluorescence in situ hybridization, 140
Tissue enclosed processing of, as a safety mea
sure, 209 preparation of, safety considerations in
managing, 213-215, 219-220 preservation of, for immunocytochemi
cal studies, 6-29 Toxic substances, risks of heating in micro
wave ovens, 210--211 Transmission electron microscopy
for examining viruses, 73-107 limitations of, 7 relative advantages of London resin
for, 44 See also Electron microscopy
Transplantation, physiology and immunology of, 237-240
Trypsin, for antigen retrieval, 57 Tuberculosis, biological hazard from
handling tissues involving, 213 Tubulitis, following with immunolabeling,
237-240 Tumor necrosis factor-related apoptosis
inducing ligand (TRAIL), 229 Tumors
drug resistance in, proteins involved in, 248
identification and grading of, 234-237 reproductive tissue, diagnosis and
monitoring of, 236
suppression of, roIes of intercellular communication proteins in, 236
Tungsten (sodium phosphotungstate), for immunonegative staining, 39-40
Tyramide signal amplification (TSA), for increasing sensitivity of in situ hybridization, 124-125, 166-168
Ubiquitin, in the cell cycle, identifying, 228
Unicellular organisms, detecting antigens on the surfaces of, 41
Unmasking, of antigens, 55-72 Uranyl acetate
safety considerations in use and disposal of, 215
toxicity of, 214
Variance, components of, 190--192 Variation
separating components of, design for, 189-192
statistical understanding of, and decision making, 188-189
Virological monitoring, with affinity labeling, 240--246
Index 267
Visualization, in light microscopy, fluorescence for, 2
Waste chemical, disposal regulations, 219 laboratory, 207-208
disposal regulations, 215 Water baths
heating in, for antigen retrieval, 69 maintenance for safety, 209-210
Water drop technique, for immunonegative staining, 40
Wax embedding for light microscopy, 16-18,88
storage and preservation, 38 safety considerations in using, 209, 214
Wax sections antigen retrieval in, with proteolytic
enzymes, 57-58 for light microscopy, 32-33
Web, Protein Reviews on the, 227 Winchester carriers, for safe transport of
liquids, 206 Working practices, safe, 205
Xylene, threshold limit value for, 216