Biomarkers 

Biochemical end points can be applied on tissue samples collected during or after all of the studies. Effects of transgene, surgical manipulation and/or compound treatment can be studied on three levels: transcription (when DNA is transcripted to mRNA), translation (when mRNA is processed into protein), and activity (how the active protein / enzyme function is modified).

Charles River provides several tools to study these different levels of biomarker expression:

• mRNA levels by qPCR
• Protein levels by Immunohistochemistry/Immunocytochemistry, Western blotting and ELISA
• Protein activity by assays
• Histology
• In vitro spectroscopy for short-lived metabolites

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qPCR

Amplification Plot

Synaptophysin mRNA is decreased significantly in aged rat cortex.

IMMUNOHISTOCHEMISTRY

Validated immunohistochemical end points include:

• Cellular proliferation with BrdU or Doublecortin
• Neuronal visualization with NeuN or MAP-2
• Dopaminergic neurons with tyrosine hydroxylase (TH)
• Striatal medium spiny neurons with DARPP-32
• Microgliosis with Iba-1 or CD11b
• Monocytes/macrophages with CD68
• Astrogliosis with GFAP
• Myelin / white matter with Myelin Basic Protein (MBP)
• Huntingtin protein with EM-48
• Iron detection with Ferritin

Immunoreactivity of myelin basic protein, a marker for white matter, in rat spinal cord

CD68 immunoreactive monocytes/macrophages in rat spinal cord after injury

Aβ immunoreactive plaques can be found in 9-month-old CVN mouse hippocampus, but not in wild-type littermates.

Tyrosine hydroxylase immunoreactive neurons in rat substantia nigra

Iba-1 immunoreactive microglia in R6/2 mouse striatum

WESTERN BLOTTING

Expression of different C-terminal APP-fragments can be analyzed from AD mouse tissue samples by Western blotting.

ELISA

Validated ELISA end points include

• Aβ1-40 (soluble and insoluble)
• Aβ 1-42 (soluble and insoluble)
• IL-1β
• TNF-α
• TGF-β
• Tau
• Phospho-Tau
• Glucagon
• Insulin

Increased amount of Aβ1-42 can be detected in plasma, CSF and tissue samples of Tg2576 mice already at 5 months of age. Gamma-secretase inhibitor DAPT significantly decreases the amount of Aβ1-42.

HISTOLOGY

Histology end points include:

• Cresyl Fast Violet
• Fluoro-Jade B
• Giemsa
• Hematoxylin & Eosin
• Luxol Fast Blue
• Perls (iron)
• TTC

Histological staining of rat brain with CFV, close-up on hippocampus

Histological staining of rat spinal cord with Giemsa

Fluoro-Jade staining can be used for identification of degenerating neurons

Hematoxylin-Eosin stain can be applied to evaluation of brain tissue damage.

IN VITRO SPECTROSCOPY

Using the Muromachi microwave brain fixation system the short-lived brain metabolites and neurotransmitters can be detected using in vitro spectroscopy.

 For more information, please contact us at askcharlesriver@crl.com.