Chromogens for HRP
DAB (3,3’-Diaminobenzidine) is typically used as a signal enhancer in conjunction with the HRP-based immunostaining systems. The dark brown end-product derived from DAB is insoluble in water and alcohol, stable and suitable for long-term storage. In addition, the end-product could be observed under a light microscope or processed with OsO4 for observation under electron microscopy. Hematoxylin, methyl green and methyl blue are the compatible counterstains. Since DAB may cause skin and bladder cancers, it is advised that personal protective equipment should be used and skin/mucosa should be avoided.
After staining with AEC (3-Amino-9-Ethylcarbazole), the positive area on tissue section changes to dark red. The end-product derived from AEC is soluble in organic solvent and cannot be stored on a long-term basis. Similar to DAB, hematoxylin, methyl green and methyl blue are some of the suitable counterstains for AEC. Glycerin gelatin should be used as the AEC mounting medium.
Chromogens for AP
Used in conjunction, BCIP (5-Bromo-4-Chloro-3-Indolyl-Phosphate)/NBT (Nitro Blue Tetrazolium) is a widely accepted chromogenic substrate used in the AP-based immunostaining systems. After exposing to AP, the substrate changes to bluish violet or black violet. The end-product derived from BCIP/NBT is insoluble in alcohol. Nuclear fast red and brilliant green are the suitable counterstains for BCIP/NBT.
- Fast Red TR Salt
Fast Red is also used for the colorimetric detection of AP. Its end-product has a rose color and is soluble in alcohol. These counterstains are used for the Fast Red chromogens: methyl green, brilliant green and soluble hematoxylin.
After staining the target antigen by IHC, a secondary stain is usually applied to provide contrast that helps the primary stain more distinct. While many of these stains show specificity for discrete antigens or cellular compartments, other stains will deliver the staining of a whole cell. Some of the most common counterstains are described as follows:
Hematoxylin, a natural dye which is extracted from the heartwood of the logwood tree, is used for cell nucleus staining. Differentiation refers to the process of using reagents (e.g. 1% hydrochloric acid HCl and alcohol) to remove the color caused by overstaining or non-specific staining on sample tissues. After running nucleolar staining (in aluminum hematoxylin) and differentiation (in HCl and alcohol), the tissue section is transferred from an acid solution to an alkaline solution (e.g. ammonia water and disodium hydrogen phosphate solution). During this process, the section will change from red brown into blue. This procedure is known as bluing.
- Hematoxylin is sub-categorized into Mayer’s Hematoxylin and Harris Hematoxylin.
- Mayer’s Hematoxylin is reddish violet and is valued for several properties: low staining time, no perception and metal membrane as well as no post-staining differentiation.
- Harris Hematoxylin is purple red, widely used in H&E staining and has these advantages: fast staining, bright color, clear nucleolar stains and well defined tissue morphology. Although metallic oxide may float on the Harris hematoxylin solution after a long period of time, filter is unnecessary before use as no perception will appear. Differentiation and bluing should be carried out after staining with Harris Hematoxylin.
- Methyl Green
Methyl green consists of metallic green microcrystals or bright green powders. It becomes bluish green when dissolved in water. This basic dye can be easily bounded with highly polymerized DNA and changes the nucleus to green. Counterstain with methyl green takes 2 to 5 min which should be followed by washing the sample, dehydration and mounting.
- Nuclear Fast Red
This counterstain will change the nucleus to red after applying to the tissue section for 2 to 5 min.
A mounting medium may be used to attach a coverslip or may itself be used to replace the coverslip. Generally, the medium selection depends on a few factors including the chemical compatibility with chromogen and counterstain as well as the preservation period.
- Neutral Mounting Medium
It usually refers to an oily substance with pH 7.0 such as neutral gum (resin). Before mounting, the sample should be treated with dimethyl benzene, transparent and dehydrated for long-term storage sections.
- Water-Soluble Mounting Medium
Popularly used in IF staining for short-term storage sections, this mounting medium usually consists of 50% glycerol.
The table below summarizes the choice of mounting medium among different enzymes, chromogens and counterstains.
|HRP||DAB||Hematoxylin, Methyl Green, Methyl Blue||Neutral|
|HRP||AEC||Hematoxylin, Methyl Blue||Water Soluble|
|AP||BCIP/NBT||Nuclear Fast Red, Brilliant Green||Neutral|
|AP||Fast Red||Hematoxylin, Methyl Green, Brilliant Green||Water Soluble|