Processing brain slices for microscopy

Today’s experiment in the lab was all about staining brain slices with Hematoxylin & Eosin (H&E) for microscopy. In the timelapse below, you can see me performing the experiment, which lasted about 3.5 hours.

This experiment was part of my current project at the university, for which the main aim is to establish the therapeutic potential of a centrally-administered peptide 35 minutes after induction of experimental stroke, by examining final infarct volume and ischaemic lesion growth from MRI images. Because the MRI images collected (DWI and T2-weighted images) do not reflect a quantifiable measure of cell death, but rather show different patterns of oedematous activity, H&E staining was performed to ensure validity of the imaging results after oedema correction.

H&E staining experiment

Timelapse: Performing the H&E staining experiment.

Detailed methods of today’s experiment

A total of 54 brain slices were stained. Paraffin-processed brain sections were immersed in histoclear, followed by immersion in a series of graded alcohols. Haematoxylin stain was then applied, followed by a series of washing and differentiation in acid alcohol. The slices were then dehydrated and stained with eosin, followed by further dehydration and finally immersion in histoclear. Stained sections were mounted using distyrene, plasticiser, and xylene (DPX) mounting medium. Basophilic structures are stained blue by haematoxylin, whereas eosinophilic structures are stained pink by eosin. Therefore, neuronal nuclei will appear blue and the cytoplasm and extracellular space will appear pink under a light microscope.