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300592 UE Basics of Neuroscience. 902557 BVO + 902558 UE Methodes and Techniques in Neuroscience. Michael Berger Center for Brain Research Introduction to slice autoradiography.

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slide1

300592 UE Basics of Neuroscience

902557 BVO + 902558 UE

Methodes and Techniques in Neuroscience

Michael Berger

Center for Brain Research

Introduction to slice autoradiography

slide2

For binding experiments, most commonly membrane preparations are used (membranes of homogenized tissue). A more ambitious, but also more time-consuming technique is the binding of radioligands to slices of intact tissue, with preserved tissue integrity. Sometimes, this technique is still described as new, but ...

slide3

For binding experiments, most commonly membrane preparations are used (membranes of homogenized tissue). A more ambitious, but also more time-consuming technique is the binding of radioligands to slices of intact tissue, with preserved tissue integrity. Sometimes, this technique is still described as new, but ...

W.S. Young & M.J. Kuhar (1979) A new method for receptor autoradiography: [3H]opioid receptors in rat brain. Brain Res. 179: 255-270

slide4

For binding experiments, most commonly membrane preparations are used (membranes of homogenized tissue). A more ambitious, but also more time-consuming technique is the binding of radioligands to slices of intact tissue, with preserved tissue integrity. Sometimes, this technique is still described as new, but ...

W.S. Young & M.J. Kuhar (1979) A new method for receptor autoradiography: [3H]opioid receptors in rat brain. Brain Res. 179: 255-270

... it‘s probably older than most of you.

slide5

Slice-autoradiography allows the semi-microscopic evaluation of binding site distribution in fixed, partially fixed, or unfixed (native) tissue. “Semi-microscopic“ means a resolution down to 50 – 10 µm, i.e. down to cellular dimensions (in mammalian tissue).

slide6

Since long, tissue slices have been obtained in good quality from fixed material, i.e. from biological tissue treated with a fixative as formaldehyde. With these fixation techniques, subcellular structures have been visualized since the times of Golgi and Cajal.

slide7

Since long, tissue slices have been obtained in good quality from fixed material, i.e. from biological tissue treated with a fixative as formaldehyde. With these fixation techniques, subcellular structures have been visualized since the times of Golgi and Cajal.

After these treatments, however, most receptors are no longer recognized by their natural ligands.

slide8

For receptor autoradiography, the tissue is only slightly fixed, or not fixed at all. To allow the preparation of thin slices, the tissue is frozen, and kept frozen in a cryostat.

slide10

Don‘t freeze the tissue by simply putting it into a – 20 °C refrigerator.

(It will look like this – freezing artefacts)

slide12

Best tissue quality needs shock-freezing.

Here the advice is: shock-freeze to -18 °C. But we go further...

slide13

Method of choice: freezing mixture with dry ice and organic solvent (mostly isopentane), kept at – 45 to – 40 °C.

slide14

Method of choice: freezing mixture with dry ice and organic solvent (mostly isopentane), kept at – 45 to – 40 °C.

Temperature must be supervised with a thermometer: If you wait too long, it will reach – 78 °C (the sublimation temperature of CO2).

slide15

To avoid freezing artefacts in the middle of the tissue, at least one dimension must be below 10 mm.

Specimens, that are successfully frozen:

For a rat brain, a 100 ml beaker will be sufficient.

slide16

To avoid freezing artefacts in the middle of the tissue, at least one dimension must be below 10 mm.

Specimens, that are successfully frozen:

For a rat brain, a 100 ml beaker will be sufficient.

For a human brain slice, a 1.000 ml jar will be necessary.

slide18

The frozen tissue is transferred to the cryostat chamber (kept at – 10 to – 20 °C) and mounted with...

slide19

The frozen tissue is transferred to the cryostat chamber (kept at – 10 to – 20 °C) and mounted with...

... a cryo-gel, mostly “Tissue-Tec® O.C.T. compound“ (from optimal cutting temperature) to ...

slide20

The frozen tissue is transferred to the cryostat chamber (kept at – 10 to – 20 °C) and mounted with...

... a cryo-gel, mostly “Tissue-Tec® O.C.T. compound“ (from optimal cutting temperature) to ...

... metal holders that can be fixed to the microtome.

slide21

The instrument holding the knife and moving the object is a microtom. To prepare frozen sections, it is kept in a kryostat.

slide22

The instrument holding the knife and moving the object is a microtom. To prepare frozen sections, it is kept in a kryostat.

The mounted tissue is moved across the knife, leaving on it the semi-thin sections (10 – 30 µm).

slide23

The frozen section is taken up with a coated glass slide by “thaw mounting“; the tissue is transformed from the frozen to the unfrozen state.

slide24

The frozen section is taken up with a coated glass slide by “thaw mounting“; the tissue is transformed from the frozen to the unfrozen state.

Coated glass-slides are available (e.g. coated with poly-lysine or with aminoalkylsilane), or can be coated by dipping into

0.5% gelatine

+

0.05% chrome alume = KCr(SO4)2 . 12 H2O.

slide25

The tissue-sections are allowed to dry on the coated slide (usually several per slide) at low temperature, but without freezing (to avoid freezing artefacts).

M Herkenham and CB Pert (1982) Light microscopic localization of brain opiate receptors: a general autoradiographic method which preserves tissue quality. J Neurosci 2: 1129-1149.

slide26

The tissue-sections are allowed to dry on the coated slide (usually several per slide) at low temperature, but without freezing (to avoid freezing artefacts)

M Herkenham and CB Pert (1982) Light microscopic localization of brain opiate receptors: a general autoradiographic method which preserves tissue quality. J Neurosci 2: 1129-1149.

Only after complete drying (e.g. overnight), the slides are transferred to cassettes and stored at – 80 °C.

slide27

For logistic reasons, multiple slides with almost identical tissue sections (i.e. from the same neuroanatomical level) must be obtained (at least one slide for total and another for non-specific binding).

slide28

For logistic reasons, multiple slides with almost identical tissue sections (i.e. from the same neuroanatomical level) must be obtained (at least one slide for total and another for non-specific binding).

Specific binding = total binding – non-specific binding

slide30

H1

H2

H3

H4

H5

G5

G1

G2

G3

G4

E2

E3

E4

E5

F1

F2

F3

F4

F5

I5

I1

slide33

The coating keeps the tissue slice during the incubation to the glass, and the radioligand diffuses freely to its binding sites.

slide34

The coating keeps the tissue slice during the incubation to the glass, and the radioligand diffuses freely to its binding sites.

The radioligand penetrates the slice immediately and binds to all receptors (not only at the surface). With the slice technique, the same kinetic constants are obtained as in suspension.

slide35

The section is brought in contact with the radioligand ...

... either by immersing the slide into a bath ...

slide36

The section is brought in contact with the radioligand ...

... either by immersing the slide into a bath ...

... or by covering the section with a droplet containing the radioligand.

slide37

After reaching saturation equilibrium, the sections are rinsed several times with fresh buffer. As final washing step, they are shortly (seconds) dipped into deionized water, and dried quickly.

Turn heating off.

slide38

Autoradiography

At best, 50% of the radiation reaches the film / screen.

slide39

Autoradiography

Less radiation reaches the film / screen from radioligand ...

slide40

Autoradiography

Less radiation reaches the film / screen from radioligand lying deeper in the tissue.

slide41

Autoradiography

The beta radiation of 3H travels in tissue only 6 µm. Therefore, increasing the thickness of the tissue beyond a certain limit (6 µm dry tissue, ~ 30 µm frozen tissue) does not result in more radiation reaching the film / screen.

slide43

The grey levels of the film / screen are evaluated by comparison with brain-mash containing known amounts of radioactivity. Alternatively, calibrated plastic strips are commercially available.

Rainbow, Biegon, Berck (1984) J Neurosci Meth 11: 231-241.

slide44

Optical Density:

OD = log (Io / I)

Io: intensity of light before passing object

I: intensity of light after passing object

slide45

Subtraction of background gives „relative OD“:

log (Io / I) - log (Io / Ib)

= log (Ib / I)

Ib: intensity of light after passing film only

We need measurements of the shades of grey (I) and of the film background (Ib); we don‘t need Io.

slide46

Problem with 3H: After soaking slices with either [3H]leucine or with [14C]leucine (a compound that distributes evenly), only the 14C-autoradiogram shows even distribution of the label. In the 3H-autoradiogram, white matter absorbes (“quenches“) a significant fraction of the (weak) radiation.

Kuhar & Unnerstall (1985) TINS Feb. 1985, 49-53.

slide47

Advantage of film: higher resolution (down to 10 µm).

Advantage of screen: shorter exposure time (a few days).

slide48

Advantage of film: higher resolution (down to 10 µm).

Advantage of screen: shorter exposure time (a few days).

Hurter & Driffield (1899) J Soc Chem Ind 18

Plots of film density (log of opacity) versus the log of exposure are called characteristic curves, or Hurter–Driffield curves.

slide49

Conventional films respond linearly over 1-2 orders of magnitude, whereas phosphor screens have a linear range of 4-5 orders of magnitude.

http://www.mchem.btinternet.co.uk

slide50

Storage phosphor radiography is a digital technique that uses photo-stimulable phosphor screens to substitute for conventional screen-film combinations. While the technique is more than 15 years old, it is only recently that technological and economic aspects of these systems have become favourable enough to envisage a more widespread application.

C. M. Schaefer-Prokop, M. Prokop (2004) Storage phosphor radiography. European Radiology 7, S3, S58-S65.

slide51

Storage phosphor radiography is a digital technique that uses photo-stimulable phosphor screens to substitute for conventional screen-film combinations. While the technique is more than 15 years old, it is only recently that technological and economic aspects of these systems have become favourable enough to envisage a more widespread application.

C. M. Schaefer-Prokop, M. Prokop (2004) Storage phosphor radiography. European Radiology 7, S3, S58-S65.

slide52

How storage phosphor works

  • Exposure of the storage phosphor screen to ionizing radiation induces latent image formation
  • During laser scanning, the BaFBr:EU+2 crystals in the screen release energy as blue light
  • and return to ground state
  • Blue light is collected and measured to form a quantitative representation of the sample.

http://www.mchem.btinternet.co.uk

slide53

How storage phosphor works

  • Exposure of the storage phosphor screen to ionizing radiation induces latent image formation
  • During laser scanning, the BaFBr:EU+2 crystals in the screen release energy as blue light
  • and return to ground state
  • Blue light is collected and measured to form a quantitative representation of the sample.

http://www.mchem.btinternet.co.uk

slide54

How storage phosphor works

  • Exposure of the storage phosphor screen to ionizing radiation induces latent image formation
  • During laser scanning, the BaFBr:EU+2 crystals in the screen release energy as blue light
  • and return to ground state
  • Blue light is collected and measured to form a quantitative representation of the sample.

http://www.mchem.btinternet.co.uk

slide55

How storage phosphor works

  • Exposure of the storage phosphor screen to ionizing radiation induces latent image formation
  • During laser scanning, the BaFBr:EU+2 crystals in the screen release energy as blue light
  • and return to ground state
  • Blue light is collected and measured to form a quantitative representation of the sample.

http://www.mchem.btinternet.co.uk

slide56

Light is emitted when an x-ray is absorbed within the phosphor screen. This light scatters multiple times off the phosphor grains before it escapes the screen. The scattering causes image blur or resolution loss. Thicker phosphor screens absorb x-rays better but cause more blurring.

http://www.sunnybrook.ca/

slide57

Light is emitted when an x-ray is absorbed within the phosphor screen. This light scatters multiple times off the phosphor grains before it escapes the screen. The scattering causes image blur or resolution loss. Thicker phosphor screens absorb x-rays better but cause more blurring.

http://www.sunnybrook.ca/

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