EAE Analysis Options

As part of our CRO services, Hooke offers a wide range of tissue collection and analysis. Of course, we are also happy to ship your samples to a lab of your choice anywhere in the world.

Hooke performs:

Sometimes our customers order analysis of all groups in advance, but we generally encourage customers to wait for initial results before deciding how much analysis to order.

We usually quote a price for your base study, together with prices for additional analysis that you can order later. This gives you flexibility to analyze only a sample of animals, choose particular groups for analysis, or even skip analysis completely if the results aren't promising. Our scientists will review your initial results with you, and can recommend the best analyses to maximize return from your study.

We generally ask for 5 days notice to schedule tissue collection or analysis (but we do our best to accommodate last-minute orders).

Histological and immunohistochemical (IHC) analysis

Histological and IHC analyses provide additional readouts of drug efficacy, independent of clinical observations. While these analyses provide valuable information about EAE severity, they must be evaluated with a knowledge of the course of EAE.

Tissue sections are typically stained with hematoxylin and eosin (H&E) and anti-MBP. Our standard analysis includes:

Other frequently performed analyses include:

Typical findings in spinal cords

EAE staining

Histology of spinal lumbar sections from EAE-induced C57BL/6 mice

Mice were induced with EAE by immunization with MOG35-55 (+PTX) for 28 days. Positive control (A, B) treated orally with FTY720 (3 mg/kg, daily) and negative control (C, D; vehicle alone, daily) tissues were collected, fixed, processed and stained with H&E (A, C) or luxol fast blue (LFB; B, D) for myelin. Vehicle treated mice have increased inflammatory infiltrates (C, closed arrows) and extensive demyelination and vacuolization (D, open arrowhead) compared to treated controls.

Scale bar = 250 µm for full size images and 50 µm for insets.

(Click for larger image.)


EAE IHC MBP staining

IHC of myelin basic protein (MBP) in spinal lumbar sections from EAE induced C57BL/6 mice

EAE was induced by immunization with MOG35-55 (+PTX) for 28 days. Positive control (left) treated orally with FTY720 (3 mg/kg, daily) and negative control (right; vehicle alone, daily) tissues were collected, fixed, processed and stained for myelin basic protein (MBP; brown). Scale bar 250 µm for full size image, 50 µm for insets.

Flow cytometric analysis

Hooke offers flow cytometric analysis of cell-surface and intracellular molecules. These analyses can help confirm or identify the mode of action of your compound.

We normally have a wide range of antibodies in stock and routinely perform 9 color analysis. We perform both surface and intracellular staining.

Most often we analyze CNS-infiltrating cells from brain or spinal cord. Spleen and inguinal lymph node cells are also frequently analyzed.

Typical flow cytometric results - CNS infiltrating cells in chronic EAE

Below are typical results from cell surface and intracellular staining of CNS-infiltrating cells from a C57BL/6 mouse with chronic EAE.

Spinal cords were collected and infiltrating cells isolated using Percoll gradient, then cells counted.

Cells were placed in culture with phorbol myristate acetate (PMA, 50 ng/mL), ionomycin (0.5 µg/mL) and brefeldin (1 µg/mL) and incubated for 4-5 hours, then washed and stained.

All cells
CD4 APC vs. CD8 PE
CD4+ cells
IL-17A PE vs. IFNγ FITC
CD4+ cells
GM-CSF PE vs. IL-10 FITC
CD4+ cells
CD25 PE vs. FOXP3 Alexa 488

Ex vivo T cell function analysis (restimulation with antigen)

Proliferation analysis (BrdU staining)

Cell proliferation can be analyzed by culturing cells in the presence of bromodeoxyuridine (BrdU), a thymidine analog. As cells proliferate in culture, the BrdU is incorporated into DNA in place of thymidine. The presence of BrdU can be detected with anti-BrdU antibodies.

Spleens or lymph nodes are collected and cell suspensions are setup in 24-well plates with multiple concentrations of antigen for 48 to 72 hours.

BrdU is added to wells for 3 to 4 hours. Cells are then collected, stained with anti-BrdU antibodies, and flow cytometry is performed.

Typical BrdU results

0.0 µg/mL MOG35-55
0.0 ug/mL MOG35-55
6.6 µg/mL MOG35-55
6.6 ug/mL MOG35-55

Please contact Hooke at or with questions or for a quotation.