tissue prep and protein assay 2012

7/25/2019 Tissue Prep and Protein Assay 2012

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Preparation of brain tissue

The rat brain


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The rat brain

Perfused rat brain


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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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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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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

tissue prep and protein assay 2012

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