tony beristain helsingor bondtalk2
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Profiling the mouse brain using fluorescent immunolabeling
Tony Beristain Ph.D.
Tissue Profiling Facility Science for Life Laboratory
KISP (Karolinska Institute Science Park)
Aims and Objectives
Producing an antibody-based protein distribution profile of the rodent brain covering a large portion of
the brain proteome
•Validation of HPA antibodies for use on rodent tissue
•Create a platform for detailed analysis of protein distribution in animal models or human (brain) tissue
•Help identifying novel key protein
Protein distribution profile in the adult CNS
Regions
Cell types
Subcellular
Organelle
Lundberg & Uhlén, Proteomics, 2010
Subcellular Protein Atlas
Mouse brain
Developing mouse
• Spatial profile– Nervous system– Peripheral organs
• Temporal profile– Midgestation (E12.5)– Late gestation (E17.5)– Early postnatal (P2)
• Cell type– Stem cells– Progenitors– Differentiating cells
Mulder et al., EJN, 2010
Neurological disorders
• Diseased brain– Animal models– Human tissue
• Changes in protein distribution/expression– Regional– Subcellular– Associated with pathological
hallmarks
Experimental Approach
Mulder et al., 2007 (Neurosci.)
Resolution 16,000 x 30,000 pixels
Fully automated IHC
Capable of processing big volumes
Speed
Efficiency
Reproducible quality
Simplicity of operation
No daily maintenance
Widely used in several hospital around the world
Building up and Optimizing our protocol
Adding steps:
Pre incubations (PBS/Ethanol/Methanol)
Washes (PBS/TrisNaCl)
Classic IHC protocol
Playing with variables:
Concentration (1°and 2° ab)
Diluting abs in DMSO/Glycerol
No. of washes
Time
Thickness of tissue
αHPA034492αHPA008176
αHPA008273
So far…
BOND IHC
Mapping up to 6 antibodies per day
Overnight run
Reproducible staining quality
Gentle to the tissue
Reduce volume of antibodies needed
Gain of time
CLASSIC IHC
3 antibodies per 1.5 days
Variable staining quality
Some sections come off during washes
High volumes of antibodies
Time demanding
Exhausting manual labor
ADVANTAGES
vs
So far…
BOND IHC
Non uniform staining
CLASSIC IHC
Uniform staining
vs
DISADVANTAGES
αTH
αTHαHPA023338
αHPA023338αHPA023338
Wishing list to Leica-Bond
• A more open software
• Menu to modify temperature in the ISH modality
• More thoughtful to non pathologists researchers.
For enquires contact:
Jan Mulder Ph.D.
Facility Manager
Tissue Profiling Platform
Science for Life Laboratory
Tomtebodavägen 23 A
Stockholm Sweden