Immunohistochemistry is widely used in Neuroscience studies and requires careful work up and inclusion of positive and negative controls to ensure the final label is specific and sensitive. If this is not done the result is in question as merely an artifact of processing and staining. Work up requires titration of the antibody concentration and a sensitive positive control. Isotype antibodies should also be run with the experiment to exclude staining caused by inter species reactivity and other non-specific binding reactions.
Polyclonal and monoclonal antibodies are available for use and have the ability to label target proteins. The limitations of these types of antibodies should be considered where a small poorly expressed protein is the target and where cross reactivity with the species of the tissue may be an issue. Pre adsorbed antibodies are available at extra cost but these are useful for multiple labeling techniques.
Antigen retrieval is required for most aldehyde fixed tissues and is essential to reveal antigens in the tissue. Fresh frozen sections can be post fixed in acetone to eliminate the need for cross linking fixatives and this can be useful when looking for fixation sensitive epitopes.
In tissues that have very low expression of the antigen of interest amplification kits using Tyramide are commercially available and this provides better signal to background staining. (for Tyramide Signal Amplification (TMA) protocal see page 24)
Fluorescence and Chromogenic Labeling
The type of label used in immunohistochemistry will be dependent on levels of expression of the protein of interest. The lower the expression and smaller the tissue entity the more sensitive the labeling needs to be. Bright fluorescent labels in the orange to green wavelengths are best for small or low expression especially if multiple labels are being applied. For strongly expressed antigens a chromogen such as DAB+/- nickel enhancement will allow for long term archiving of slides. Make sure your final chromagen compliments any counterstain color.
A recommended setup if you want to observe multiple proteins is:
DAPI/Alexa350 + Alexa488/GFP + Alexa594 + Alexa647
(Look out for crosstalk between Red and Far Red channels)
Another 4 colour combination is:
DAPI/Alexa350 + Alexa488/GFP + Alexa555 + Alexa647
(Look out for crosstalk between Green and Red channels)