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