Drug Localization in Tissues and Cells. Receptor Microscopic Autoradiography.
Author: Walter E Stumpf.
ISBN 0-9740515-0-0. Library of Congress Control Number 2003105179.
IDDC-Press, Chapel Hill, NC. July 2003. 170 pages with color plates. $ 164.--.

The book is available through the University of North Carolina
Bookstore in Chapel Hill (UNC Student Stores, Chapel Hill , NC 27599 ).

 

Cover Text:
Where do drugs act in the body? Which are the targets for desired therapeutic effects,
for other depositions that may lead to side effects or engender toxicity? What are the
routes of drug distribution, metabolism, and excretion? How much of a certain drug is
bound to receptors, what is the duration of binding, and how does this relate to action?
In order to answer such important questions, detailed information about in vivo sites of
drug deposition is essential. Techniques with high resolution and high sensitivity are
required. Since organs and tissues are composed of different cell types with different
functions, cells and specific cell populations need to be properly identified and
diagnosed. And since low numbers of molecules can act at specific cells and receptors,
their detection must be made possible. For that, microscopic resolution is necessary.
While current non-invasive scanning methods, as well as whole body autoradiography and
conventional radioassay-HPLC with excised organs or pieces of them, are valuable to gain
insight into drug distribution rates in blood, metabolic organs, excretory fluids, and
other high capacity/low specificity sites, they lack both resolution and sensitivity.
These expedient methods are unfortunately unable to identify high specificity/low
capacity receptor sites of drug action. Likewise, results from in vitro techniques,
although helpful, cannot be used as a substitute for receptor binding of drugs under in
vivo conditions. Receptor Micro Autoradiography, as detailed in this book, was developed
for the microscopic tissue localization of drugs, autacoids and xenobiotics,
non-covalently bound molecules in general. The method is based on the use of radiolabeled
compounds with high specific activity in conjunction with nuclear emulsion, and with
tissue preparation that preserves in situ conditions. Cellular-subcellular detail can be
viewed, associated radioactivity characterized and quantified, and time- and dose-related
data of different compounds compared. This manual contains instructions on experimental
design, execution, and evaluation of autoradiograms. Precise localization is documented
to provide important clues for function and to serve as a guide for further
histochemical, biochemical, functional, and clinical follow-ups for the development of
new therapeutic applications.
Receptor Micro Autoradiography, with its high diagnostic and predictive values, is an
indispensable tool for drug research and development.

Table of Contents

  1. Introduction 1
    1. General Considerations 1
    2. History 5
      1. Macro Au to radiography—Apposition (Sandwich) Techniques 5
      2. Receptor Microscopic (Micro) Autoradiography 6
  2. Design and Introduction of Experiments 8
    1. Planning 8
    2. Pilot Studies 10
    3. Introductory Experiment to establish the Autoradiographic Procedure 1
  3. Emulsion Coating of Slides 13
    1. General Considerations 13
    2. Dipping Procedure 13
    3. Control of Emulsion Background I6
      1. Initial Background Count 16
      2. Experimental Background Count 16
  4. Radiolabeled Compounds 18
    1. General Considerations 18
    2. Choice of Radioisotope 18
      1. Tritium (3H) I8
      2. Radiocarbon (14C) 19
      3. Radioiodine (125I) 20
    3. Purity of Labeled Compound 20
    4. High Specific Activity 20
    5. Dose and Route of Application 21
    6. Characterization of Radioactivity in Tissues and Specificity of Binding 22
  5. Procurement of Tissue 23
    1. Arrangements 23
    2. Dissection and Placement of Samples 23
    3. Samples for Parallel Alternate Studies 24
  6. Freezing of Tissues, Freeze-Mounting, and Storage 26
    1. Freezing and Control of Ice Crystal Formation 26
    2. Freezing Procedure 27
    3. Freeze-Mounting 28
    4. Modifications of Freezing Procedure 31
  7. Frozen Tissue Sectioning and Thaw-Mounting 32
    1. Transfer of Frozen Specimen from Storage to Microtome-Cryostat 32
    2. Orientation of Specimen and Trimming 32
    3. Cutting Temperature 33
    4. Frozen Sectioning 35
    5. Frozen Section Thaw-Mounting on Emulsion-Coated Slide 37
    6. Consideration of Modifications 38
  8. Photographic Exposure and Development 40
    1. Storage of SIides in Desiccator Boxes 40
    2. Exposure Time 41
    3. Photographic Development and Fixation 43
    4. Histological Fixation 45
  9. Staining 47
    1. General Considerations 47
    2. Selecting the Stain 48
    3. Preparing and Applying the Stain 48
      1. Methylene Blue—Basic Fuchsin Stain (MBBF) 49
      2. Methyl Green—Pyronin Stain (MGP) 49
    4. Testing and Stability of the Stain 51
  10. Evaluation of Autoradiograms 52
    1. Understanding Localized Radioactivity 52
    2. Relationships Between Silver Grains and Tissue Structures 53
    3. Reproducibility and Authenticity of Results 54
    4. Problems of Interpretation of Results 54
    5. Importance of Pictorial Evidence 56
  11. Quantification 57
    1. General Considerations 57
    2. Special Considerations 59
      1. Identification of Cell Types 59
      2. Cell Compartment Size and Density of Silver Grains 60
      3. Tissue Self-Absorption of Radiation 60
      4. Selective Evaluation of Labeled Cells and Compartments 60
      5. Number of Cells Counted 61
      6. Subtraction of Background Counts 61
      7. Characterization of Radioactivity Associated with Certain Tissue Structures and Compartments 62
    3. Hierarchy of Receptor Binding in Target Tissues 63
    4. Estimation of Number of Molecules 64
  12. Colocalization—Combined Autoradiography and Immunohistochemistry 66
    1. General Considerations 66
    2. Procedure 67
      1. Fixation of Exposed Slides Prior to Auto radiographic Development 67
      2. Autoradiographic Development 67
      3. Immunocytochemical Staining 6
  13. In Vitro Experiments 70
    1. General Considerations 70
    2. Diffusion 70
    3. Handling Tissue Slices 71
    4. Incubation 72
    5. Cell Suspensions 73
      1. Spreads 73
      2. Pellets 73
  14. Artifacts 74
    1. General Considerations 74
    2. Multiple Sources of Possible Artifacts 74
    3. Artifacts Related to In Vitro Incubation 77
  15. Controls 78
    1. Controls Against Positive Chemography and Mechanical Artifacts 78
    2. Controls Against Negative Chemography 78
    3. Controls Without Radioactivity 79
    4. Competition Controls for Characterization of Localized Radioactivity 80
    5. External Positive "Controls" 80
    6. Control of Thaw-Mounting by Dry-Mounting 81
  16. Alternate Procedures for Diffusible Compounds 83
    1. General Considerations 83
    2. Dry-Mounting of Frozen Sections 83
    3. Dry-Mounting of Freeze-Dried Sections 84
    4. Problems with Hard and Spongy Tissues 88
      1. Frozen Section Thaw-Mounting 88
      2. Frozen Sectioning After Backing with Scotch Tape or Plastic Foil 89
      3. Frozen Sectioning of Acrylamide-Infiltrated Tissue 90
    5. Regional `High Resolution' Autoradiography 91
    6. In Vitro Labeling Autoradiography 92

Addendum 93

  1. Examples of Autoradiograms 95
    Plate 1. 3H-Mesobilirubinogen, Liver 98
    Plate 2. 3H-Estradiol, Uterus, Brain 100
    Plate 3. Stria Terminalis, Estrogen Target Neuron Circuit, Allocortex-Brainstem-Core (ABC) Circuitry 102
    Plate 4. 3H-Progesterone, Uterine Cervix; 3H-Org 2058, Brain 104
    Plate 5.3H-1,25(OH)2Vitamin D3, Brain, Spinal Cord 106
    Plate 6. 3H-1,25(OH)2Vitamin D3, Pituitary 108
    Plate 7.3H-1,25(OH)2Vitamin D3, Parathyroid, Thyroid 110
    Plate 8.3H-1,25(OH)2Vitamin D3, Heart 112
    Plate 9.3H-1,25(OH)2Vitamin D3, Spleen 114
    Plate 10.3H-1,25(OH)2Vitamin D3, Adrenal 116
    Plate 11.3H- 1,25(OH)2Vitamin D3, Stomach 118
    Plate 12. 3H-1,25(OH)2Vitamin D3, Pylorus, Duodenum 120
    Plate 13. 3H-1,25(OH)2Vitamin D3, Small Intestine 122
    Plate 14. 3H-1,25(OH)2Vitamin D3, Pancreas 124
    Plate 15.3H-1,25(OH)2Vitamin D3, Testis, Kidney 126
    Plate 16. 3H-Oxa-Calcitriol (OCT), Bone 128
    Plate 17. 3H-Oxa-Calcitriol (OCT), Cancer Xenograft, Skin 130
    Plate 18. Vitamin D Receptor "Drug Homunculus" 132
    Plate 19. Vitamin D (Soltriol) Biological Role 134
    Plate 20. 3H-Oxa-Calcitriol (OCT), Whole Body Autoradiograms 136
    Plate 21. Microtome-Cryostats 138
  2. Lessons Learned 141
  3. Chronology of Development of Techniques, Applications, and Discoveries 145
    1. Highlights 145
    2. Timeline of Development of Method, Related Discoveries, and New Concepts 147
  4. References for Autoradiography of Diffusible Compounds 155
    1. Selected Publications by the Author 155
    2. Additional References 166
  5. About the Author 169

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