Methods and Applications of Labeling Techniques

1
Methods and Applications of Labeling Techniques

• Elizabeth Muir and Lisa Thomas
• Neuroscience 405
• Student Taught Class
• December 3, 2003

2
Immunocytochemistry Principle and Definitions

• Based on the high specificity of the
  antigen-antibody reaction
• Consist of the detection of an antigen present in
  histological or cellular structures by
  application on tissues or cells of its specific
  antibody or antiserum
• Two families used
• Polyclonal and monoclonal antibodies
• Primary antibody
• The antibody used to recognize an antigen in the
  tissues
• Secondary antibody
• The antibody used to recognize the primary
  antibody

3
Immunocytochemistry - Applications

• Localization of neurons synthesizing a specific
  neurotransmitter
• Localization of receptors of a neurotransmitter
• Detection of antigen-antibody complex
• Reasons why secondary antibodies are labeled
  instead of primary antibodies
• Methods for labeling

4
In situ Hybridization - Principles

• Detection of a specific nucleic acid sequence in
  cells on histological sections or in cultured
  cells
• Based on the capacity of all nucleic or
  ribonucleic acids sequences to bind to a
  complementary sequence
• In situ hybridization relates to detection of
  messenger RNA detection made possible through
  use of a probe that corresponds to a sequence of
  nucleic acids complementary to DNA or RNA that is
  to be detected

5
In situ Hybridization - Application

• Technique that allows visualization of gene
  transcripts and localization of potential site of
  synthesis for protein or peptide coded by a gene
• Nerve cells
• Localize neurons that express a gene coding for a
  neurotransmitter, for a synthesis of degradative
  enzyme of a specific NT, or for a receptor for a
  NT

6
In situ Hybridization - Probes

• Double-stranded DNA
• Single-stranded DNA
• Single-stranded RNA
• Oligonucleotides

7
In situ Hybridization - Markers

• Two major families are used
• Radioactive labels
• Non-radioactive labels
• Disadvantages of radiolabeled probes
• Safety
• Limited utilization time
• Limited spacial resolution due to scattering of
  emitted radiation
• Advantages of non-radiolabeled probes
• Stability
• Give rapid results
• Display high levels of cellular resolution

8
In situ Hybridization - Detection

• Radioactive probes
• Low resolution signal can be obtained by placing
  tissue or cells mounted on slides in contact with
  X-ray film for overnight exposure
• This allow one to control efficiency of reaction
• Non-radioactive probes
• Detection of hybrids is performed by
  immunocytochemistry

9
Expression of Tryptophan Hydroxylase, Connexin
36, and Cytochrome Oxidase in the Raphe Obscurus
of the Developing Rat

• Lisa Thomas
• Medical College of Wisconsin
• Department of Cell Biology, Neurobiology, and
  Anatomy
• SPUR 2003

10
Sudden Infant Death Syndrome

• SIDS is a common cause of infant mortality
  between 1 week to 1 year of age, especially
  between 1 and 4 months of age1.
• Triple-Risk Model2
• Vulnerable infant
• Critical developmental period
• Exogenous stressor

• Liu, Q. Wong-Riley, M. J. Appl. Physiol. 2002,
  92, 923-934.
• Filiano, JJ. Kinney, HC. Biol. Neonate. 1994,
  65, 194-197.

11
Pre-Bötzinger Complex (PBC)

• The PBC is involved in rhythm generation, pattern
  formation, and respiratory modulation1.
• Previous studies involving cytochrome oxidase
  have indicated two potential critical periods
  during postnatal development of the rat.

1. Wong-Riley, M. TINS 1989, 12, 94-101.
12
CO in the rat PBC

• Two critical periods of low CO activity
• PND3-4
• PND12
• Increased activity of inhibitory
  neurotransmitters and their receptors decreased
  activity of excitatory neurotransmitters and
  their receptors

Liu, Q. Wong-Riley, M. J. Appl. Physiol. 2002,
92, 923-934.
13
Raphe nuclei

• Serotonergic neurons have become a focus of
  interest1.
• Arcuate nucleus in human
• Raphe nuclei in rat
• Chemosensitivity for detection of CO2 2
• Raphe obscurus (ROb)

• Kinney, HC. Filiano, JJ. White WF. J.
  Neuropathol. Exp. Neurol. 2001, 60, 228-47.
• 2. Richardon, GB. Wang, W. Tiwari, J.
  Bradley, SR. Respiration Physiology 2001, 129,
  175-189.

14
Hypothesis

• The raphe nuclei being related to respiratory
  control, sleep, and arousal will have a
  developmental trend similar to that of the PBC
  and other brain stem respiratory nuclei.

15
Experimental Design and Methods

• Rat brain stems from 13 different postnatal days
  were studied.
• PND 0, 1, 3, 4, 5, 7, 10, 11, 12, 13, 14, 16, 21
• 25 µm sections of frozen brain stem tissue
  (medulla and lower pons) were cut using a sliding
  microtome.
• Sections were then mounted on slides.

16
Cytochrome Oxidase Histochemistry

• As the terminal enzyme in the electron transport
  chain, CO provides information on local tissue
  energy metabolism, which is tightly coupled to a
  neurons functional activity1.
• Sections were incubated in a solution of 0.1 M PB
  with 4 sucrose, DAB, and cytochrome c at 37 C
  in the dark for exactly 3 hours.
• Sections were then rinsed in cold 0.1 M sodium
  phosphate buffer, mounted on slides, dehydrated,
  and cover-slipped.

1. Wong-Riley, M. TINS 1989, 12, 94-101.
17
Immunohistochemistry

• Method of labeling certain proteins in cells
• Tryptophan hydroxylase (TPH) 1400
• Connexin 36 (Cx36) 11000
• 3 day process
• Day 1
• Cutting, mounting, and incubation in blocking
  solution overnight at 4 C
• Blocking solution prevents nonspecific labeling
  of unwanted proteins
• Day 2
• Incubation in 1 antibody for 4 hours at room
  temperature, then overnight at 4 C
• 1 antibody recognizes and binds to specific
  amino acid sequences of a protein

18
Immunohistochemistry

• Day 3
• Incubation in 2 antibody for 4 hours at room
  temperature
• 2 antibody binds to 1 antibody
• Rinses in PBS and APB
• Incubation in 3 solution for 5 minutes at 37 C
• The 2 antibody is complexed to horseradish
  peroxidase (HRP), which catalyzes a reaction with
  the H2O2 to oxidize the DAB, which then stains
  the sections
• Rinses in APB (to stop the reaction), PBS, and
  DDW
• Dehydration and cover slipping

19
Optical Density

• Optical densitometric measurements of reaction
  product of TPH were performed with a Zeiss Zonax
  MPM 03 photometer, a 25X objective, and a 2
  µm-diameter measuring spot. White (tungsten)
  light was used for illumination, and all lighting
  conditions were held constant. The white matter
  was set as the background for each section. In
  each brain stem, 216-336 neurons in the raphe
  obscurus were read.

20
Cytochrome Oxidase
A general increase in CO activity was observed
from PND0 to PND21. However, a dramatic
decrease was observed at PND12.
Qiuli Liu, Ph.D. unpublished data
21
Connexin 36 (Cx36)

• Associated with electrically-coupled neurons
• Formation of the connexon
• 6 connexins make up 1 connexon 2 connexons make
  up an ion channel in the gap junction
• Electrically-coupled neurons send synchronous
  signals and can have widespread effects1
• Presence in raphe obscurus
• Ventrolateral surface of medulla analogous to the
  arcuate nucleus in human brain stem

1. Deans, MR. Gibson, JR. Sellitto, C.
Connors, BW. Paul, DL. Neuron 2001, 31, 477-485.
22
Cx36 in ROb
23
Tryptophan Hydroxylase (TPH)

• Rate-limiting enzyme in serotonin (5-HT)
  biosynthesis1
• Marker of serotonergic neurons
• Important in chemoreception2
• Presence in raphe obscurus
• Developmental differences observed through
  optical density

• Jonakait, GM. Schotland, S. Ni, L. Brain Res.
  1988, 473, 336-343.
• 2. Richardon, GB. Wang, W. Tiwari, J.
  Bradley, SR. Respiration Physiology 2001, 129,
  175-189.

24
TPH in the ROb
Plt0.001 in Tukeys Studentized Range Test
25
TPH in the ROb
PND 5
PND 7
PND 11
PND 12
PND 16
All photographs taken with SPOT digital camera at
10X (left) or 40X (right) objective
26
Conclusions

• TPH, Cx36, and CO all show differences in
  activity in the ROb throughout development
• CO activity generally increases but exhibits a
  sharp decrease at PND12
• The number of cells labeled with Cx36 generally
  decreases with sharp dips at PND3 , PND10, and
  PND13

27
Conclusions

• The level of TPH activity was varied with the
  highest level of activity at PND5 and lowest at
  PND21
• The peak at PND11 and decrease at PND12 in TPH
  activity in the ROb is comparable to the pattern
  of CO activity in the ROb

28
Postnatal changes in cytochrome oxidase
expressions in brain stem nuclei of rats
Implications for sensitive periods

• Qiuli Liu and Margaret T.T. Wong-Riley
• Department of Cell Biology, Neurobiology, and
  Anatomy
• Medical College of Wisconsin

29
Fig. 1. Low-magnification photomicrographs
showing the locations of the brain stem nuclei
examined in the present study. Sections from rats
of postnatal day (P) 21 were reacted for
cytochrome oxidase (CO) histochemistry. B
nucleus raphe obscurus (ROb) and medial
vestibular nucleus (MVe).
30
Fig. 2. Neurons in the rat brain stem nuclei
histochemically reacted for CO at P21. ROb (D)
31
Fig. 3. Optical densitometric measurements of CO
reaction product during postnatal development in
ROb (D). Values are means SD. ANOVA of each
nucleus showed significant differences among ages
(P lt 0.01). Tukey's Studentized range test
between successive age groups P lt 0.05 P lt
0.01 P lt 0.001 (significance between one age
group and its adjacent younger group).
32
Conclusions

• Cytochrome oxidase is a good marker for neuronal
  activity.
• The raphe obscurus of the rat brain stem exhibits
  a general increase in CO activity with a plateau
  at PND2-PND3, and a dramatic decrease at PND 12.
• This is possibly due to a transient imbalance
  between excitatory and inhibitory drives.
• The brain stem operates at a markedly lower level
  of metabolic activity at PND12. This could be a
  critical period during which an animal may be
  less able to overcome the effect of exogenous
  respiratory insults.
• If such critical periods also exist in humans,
  and exogenous stressors are introduced to a
  vulnerable infant, SIDS may result.