Methods in Spinal Cord Neuromorphology

Immunohistochemistry


 

Mebrane receptors, intracellular proteins and many other macromolecules act as antigenes. Their presence or absence, their localization and distribution in tissue sections can be determined by antigene-antibody interactions. The method was first described  by Albert H. Coons and his colleagues in 1941. Antigens can be labelled by enzimes, fluorescent dyes or radioactive elements. Direct and indirect techniques can be distinguished. When the direct method is used, the labelled antibody reacts with the antigen. Indirect method involves an unlabelled primary antibody raised against the tissue antigen, and a labelled secondary antibody against the primary one.

 

Direct and indiret methods in immunohistochemistry

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In the field of conventional immunohistochemistry biotinylated secondary antibodies with HRP-ExtrAvidin conjugate binding to the biotin or peroxidase labelled secondary antibodies are used in our lab. In both cases a water-insoluble dark brown precipitate is produced by the peroxidase enzyme from diamino-benzidine (DAB) in the presence of H2O2 exactly where the antigen in the tissue is. Due to this immunostaining antigen becomes identifiable with light microscope.

 

HRP-ExtrAvidin conjugate and HRP-labelled secondary antibody: the production of the precipitate

(HRP: horseradish-peroxidase)

 

Dark brown colour indicates the presence of CART immunoreactive

fibers in the lamina I of the rat spinal dorsal horn.

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Multiple fluorescent labelling allows simultaneous identification of different antigens. Primary antibodies are raised in different host animals and the species-specific secondary ones are conjugated with fluorescent dyes. Confocal laser scanning microscope is used to determine if the antigens co-localize or not.

 

Three different antigens identified simultaneously with three different fluescent dye-labelled secondary antibodies

 

  

Triple fluorescent labelling in the lamina I of the rat spinal cord. (green: synaptophysin, red: CART, blue: substance P)

The origin of mixed colours - yellow, cyan, magenta and white.

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The combination of multiple fluorescent labelling with electronmicroscopy is a further possibility. After the sections have been analized with confocal microscope, they are processed for electronmicroscopy.

 

 

Fluorescent dye and HRP labelled secondary antibodies are used against the same primary one.

DAB precipitate is detected with electronmicroscope.

 

 

Virtual confocal image and electronmicrograph of the same section.

Antibodies outlining the cell membrane in green and a tracer-labelled terminal in red. The electronmicrograph

confirms that the terminal duoble-labelled with fluorescent dye and DAB forms synapse on the cell.

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In our lab retrograde tracers, first of all Cholera-toxin β-subunit are mainly used for labelling of projection neurons in lamina I and III-V of the rat spinal dorsal horn. As these cells project with numerous collaterals to different areas of the brainstem (caudal ventrolateral medulla, lateral parabrachial nucleus, periaqueductal gray) and less importantly to the thalamus, the majority of  the projection neurons in the spinal cord can be labelled with retrograde tracers administered into any of the target areas mentioned above. Usually a conventional HRP-DAB immunoreaction is carried out to check the injection site, while spinal cord sections are processed for multiple immunofluorescent labelling.

 

The injection site in the brainstem,  the retrogradelly labelled neurons in the spinal cord and triple fluorescent

immunostaining of projection cells (retrograde tracer: blue, NK1-receptor: green, CART: red).

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