![]() Recently a new magenta-colored HRP chromogen with a sensitivity comparable to DAB has been described (Lohse 2016). While they contrast well to DAB they are not optimal when hematoxylin is used as a counterstain ( Petersen 2009). For automated staining systems, this requires onboard mixing of the Fast Red reagents or alternatively the staining is paused until the user can supply a freshly mixed Fast Red solution to the system.Īn alternative red chromogen, 3-amino-9-ethylcarbazole (AEC) exists for the HRP enzyme system, but this is not compatible with other peroxidase substrates due to poor color contrast between reddish-brown AEC and brown DAB ( Nemes 1987).īlue HRP substrates have previously been described. ![]() Third, Fast Red chromogen must be used within 30 minutes of mixing. ![]() This includes the DAB stain which becomes darker after incubation with Fast Red. Second, the Fast Red diazonium salt can react with a range of other substances present in the tissue section. First, Fast Red stains rapidly dissolve in ethanol and most organic mounting media. However, there are drawbacks to the AP substrate system. Both colors contrast well to the commonly used blue hematoxylin nuclear stain. No quenching of enzymatic activity is required between the two stains as the two enzymes do not function with each other’s substrate. Typically, brown and red visualization is performed using the enzymes horseradish peroxidase (HRP) and alkaline phosphatase (AP) and the substrates diaminobenzidine (DAB) and Fast Red, respectively (Malik 1982). Additionally, the visualization of the antigens is performed using different enzymes in the visualization steps. To reduce the possibility of cross-staining between the two targets, antibodies derived from different species, usually rabbit and mouse, are commonly used (van der Loos 1993). In these cases, staining the same tissue section for two antigens is a useful diagnostic tool. The localization of different antigens in relation to each other can in some cases also be important for a diagnosis. In pathology, using immunohistochemistry (IHC), this is particularly important when the biopsy is small and only a few different tests can be run on the available tissue. Allow the slide to dry at room temperature and examine under microscope.A concern with many diagnostic procedures today is to get enough information from limited sample sizes to reach an informed diagnostic decision.Without disturbing the slide, flood the distilled water and wash until the thinner parts of the film are pinkish red.A metallic sheen (or green ‘scum’) should appear on the slide if mixing is appropriate. Add approximately equal amount of buffered water (pH 6.5).The undiluted stain fixes and partially stains the smear. Cover the blood film with undiluted staining solution.Place the air-dried smear on the slide staining rack, smear side facing upwards.Prepare a film of blood or bone marrow on a microscopic slide and allow to air dry.Potassium dihydrogen phosphate, anhydrous = 0.663 gmĭisodium hydrogen phosphate, anhydrous = 0.256 gm The dye may be purchased as a powder which is then mixed to methanol or a ready-made solution may be obtained. The neutral components of the cells are stained by both components of the dye, producing variable colors. Methylene blue stains acidic cellular components such as nucleic acid and basophilic granules in varying shades of blue. Eosin stains the basic components such as hemoglobin and eosinophilic granules an orange to pink color. When diluted in buffered water, ionization occurs. Eosin Y is an acidic anionic dye and methylene blue is basic cationic dye. However, fixation helps to reduce water artefact that can occur on humid days or with aged stain. As the Wright stain is methanol based, it doesn’t require a fixation step prior to staining. Wright’s stain is a polychromatic stain consisting of a mixture of Eosin and Methylene blue. The stain distinguishes easily between the blood cells and hence became widely used for performing differential WBC counts and evaluate the morphology of blood cells. Wright’s stain is named for James Homer Wright, who devised the stain in 1902 based on a modification of Romanowsky stain. It is also used for staining bone marrow aspirates, urine samples and to demonstrate malarial parasites in blood smears. Wright’s stain is a type of Romanowsky stain, which is commonly used in hematology laboratory for the routine staining of peripheral blood smears.
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