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Preparation of brain tissue
The rat brain
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Preparation of brain tissue
The rat brain
Perfused rat brain
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Preparation of brain tissue
The rat brain
Perfused rat brain
Sections
-
Histology (Cresyl violet/H&E staining)-
Immunohistochemistry (Proteins)-
In situ hybridization (mRNA)
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Cryostat
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Cryostat
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Histology & microscopy
Stain sections
Control Stress
Induction of cFOS expression in rat brain
following exposure to psychological stress
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Preparation of brain tissue
The rat brain
Sections
Dissection- Freehand- Micropunch- LCM
Perfused rat brain
}
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Freehand brain dissection
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Cerebellum Cortex
Olfactory
Bulbs
Rat Brain: Top View
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Rat Brain: Bottom View
Brainstem
Hypothalamus
Olfactory
Tubercules
Amygdala
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Olfactory Tubercule removal
OlfactoryTubercules
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Frontal Cortex removal
FrontalCortex
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Cut to the Hypothalamus
Hypothalamus
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Striatum and cortex
Striatum
Cortex
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Striatum removal
Cortex
Striatum
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Hypothalamus removal
Hypothalamus
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Separation of middle section
Amygdala
Brainstem
Cerebellum
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Halving of middle section
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Midbrain removal
Hippocampus
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Hippocampus removal
Hippocampus
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Cortex turned over
Amygdala
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Amygdala removal
Amygdala
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Cerebellum removal
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Micropunch brain dissection
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Step 1: Prepare thick brain sections in
a cryostat or suing a brain matrixBrain matrix
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Laser capture microdissection
(LCM) of brain tissue
LCM enables: Isolation of discrete brain regions/subregions
Isolation of single cells from tissues (neurons/astrocytes/microglia)
Dissected material can be analysed by a variety of techniques
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Preparation of brain tissue
The rat brain
Sections
Dissection- Freehand- Micropunch- LCM
Tissue storage
Tissue processingPerfused rat brain
Storage- Freeze on dry ice- Store in RNAlater- Store tissue slices inKrebs + 10% DMSO
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Tissue processing-
Waking up tissue slices stored in Krebs/10% DMSO to
perform functional studies (eg) NT release- Thaw quickly, remove Krebs DMSO and wash 2-3 times before using tissueslices for functional analysis
-
-
Protein analysis- Homogenise tissue in an appropriate buffer solution containing protease
inhibitors (and sometimes phosphatase inhibitors).
- Various centrifugation steps may be required to isolate particular cellularfractions (eg) Nuclear, Mitochondrial, Cytosolic, Membrane
-
RNA analysis-
Homogenise tissue in an appropriate buffer solution and extract RNA fromtissue using an RNA extraction kit.
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Brain proteins
Brain cells achieve their specialized activities by
expressing hundreds of specific proteins, includingtranscription factors, enzymes, transporters,
structural proteins and receptors.
Therefore it is very important to study normal and
abnormal expression of various proteins in order to
delineate their physiological functions.
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How does one express the concentration of
molecules in brain tissue
Per wet weight of brain tissue
or
Per mg of protein *
*More accurate and you can equalize for total protein prior toperforming your analysis
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Methods for measuring total protein Measurement of the protein's intrinsic UV absorbance at
280nm
Absorbance280 Protein solution be free of other UV absorbing substances
Measurements are made in a quartz cuvette.
Methods which generate a protein-dependent color change
Lowry method
Biuret method
Bradford dye binding method
Under strongly acid conditions, the Coomassie Brilliant Blue G250 dye ismost stable as a protonated red form.
Upon binding to protein it is most stable as an unprotonated, blue form.
The dye reagent reacts primarily with arginine residues and less so withhistidine, lysine, tyrosine, tryptophan, and phenylalanine residues.
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1. Standard curve:
A series of standard solutions, each with a different, knownconcentration of protein
Standards should be prepared in the same medium (buffer) as
the samples have been prepared in
Protein standards:
Bovine serum albumin
Immunoglogin G (IgG - gamma globulin).
2. Determining the protein concentration in unknown tissue
samples
Tissue samples are diluted so as the protein concentration fallswithin the range of the standard curve
You can determine the protein concentration of each sample by
reading it of the standard curve.
Approach to determining the protein concentration of a tissue sample