What is Regressive Staining 4. Similarities Between Progressive and Regressive Staining 5. Progressive staining is a technique that allows the tissue in the staining solution just long enough to reach the desired endpoint. Therefore, frequent monitoring of stain quality is needed to determine the completion of staining. The staining intensity is controlled by the immersing time. For these two haematoxylings, a staining time of minutes is used commonly in progressive staining. Generally, progressive haematoxylins are less concentrated.
Hence, they slowly and selectively stain chromatin. In progressive staining, haematoxylin primarily stains the chromatin to the desired intensity. Thus, it does not require differentiation in dilute acid alcohol to pull out the excess stain. Regressive staining is a more rapid staining technique in which the tissue is deliberately over stained until the dye saturates all tissue components.
Then the tissue is selectively de-stained until it reaches the correct endpoint. De-staining step is called differentiation.
Differentiation is done to remove excess stains. Usually, it is done using dilute acid alcohol. Harris haematoxylin is the popularly used type of haematoxylin. This requirement is influenced by the natural pH of the local tap water. Nuclei that are understained with hematoxylin or over-differentiated and overstained with eosin also appear pink.
The pH of the eosin solution is monitored. It is kept close to pH 5. The addition of a couple of drops of acetic acid can be used as a convenient means of lowering pH. No attempt is made to monitor the pH of eosin. When staining intensity falls away the solution is replaced carry over of alkaline tap water can cause the pH of eosin solutions to rise. Sections are thoroughly dehydrated before being placed in xylene for clearing. Sections are sometimes rushed through alcohol to xylene.
Clearing in xylene contaminated with water can result in the presence of tiny water droplets in the tissue that are seen microscopically as opaque areas lacking detail. The coverslip is always applied before the section has a chance to dry and a high-quality mountant is used.
The long-term storage qualities of the mountant must be known because crystals can appear in poor quality mountant,sometimes after a long period months or years. Sections are allowed to partially dry before the coverslip is applied causing some nuclei to appear black. Mountant chosen based on price alone may develop crystals during long-term storage and coverslips may lift. This reference document is presented as a service to health care professionals by Leica Biosystems and has been compiled from available literature.
Although every effort has been made to report faithfully the information, Leia Biosystems cannot be held responsible for the correctness. This document is not intended to be, and should not be construed as medical advice. For any use, the product information guides, inserts and operation manuals of the various drugs and devices should be consulted. Leica Biosystems and the editors disclaim any liability arising directly or indirectly from the use of drugs, devices, techniques or procedures described in this reference document.
Geoffrey Rolls is a Histology Consultant with decades of experience in the field. With more than 25 years of experience, she is a guest speaker at histology and cytology meetings around the country.
She is a technical author for Media Lab, publishing a variety of technical courses and sharing best practices in histology. We are looking for more great writers to feature here. Send us a submission and we'll be in touch! Get more Knowledge Pathway content like this delivered directly to your inbox.
Unsubscribe at any time. Produtos em Destaque. Saiba mais. Reagent Alcohol, Isopropyl Alcohol Saiba mais. Service Back Patologia digital.
Patologia Digital Back. Todos Shop. Apply for self-reported educational credits. Note the balanced coloration in this section of skin. The nuclei are stained purple, while the cytoplasmic components are pink. Note the balanced coloration in this section of colon. The microvilli on the columnar epithelium are very crisp. Photo credit: Stanley Hansen. The red blood cells in this placenta section represent the bright coloration that may be experienced with this simple stain.
Note the mitotic figures, indicating a rapidly growing tumor. Nuclear irregularity and chromatin clumping is also noted in the cells. Photo credit: Frontalcortex. Changes in these fibers suggest disease processes that may not otherwise be detected. Note the intensity of the RBCs as compared to the pink of the cytoplasm of the surrounding cells. The red arrow indicates a malignant nucleus with very little cytoplasm. The black arrow illustrates a normalized acute inflammatory cell, similar in size to the RBCs on the previous image.
Note how much larger the malignant cell is compared to the normal nuclei on the previous image. Even the red blood cells appear muted. Less time in hematoxylin OR additional time in eosin can provide a better balance. Additional time in hematoxylin would make these nuclei much easier to see. These samples, stained with the same protocol, are very different in appearance due to cellularity. Use Accurate Timing. Each step in the staining protocol is accurately timed.
Even macroscopically, the inconsistency of the stain can be seen. Regularly Monitor Quality. Control slides are regularly stained to monitor stain quality. Placenta is another specimen type that can be used as a useful control. Standardize Staining Conditions.
One of the benefits of using an automated staining instrument is that agitation, wash and drain times are consistent. Providing other variables are properly controlled, this will ensure good, consistent results.
Ensure Complete Dewaxing. Slide dewaxing is optimized. This is due to incomplete wax removal prior to staining. Renew Reagents Regularly. This section shows poor quality, muddy hematoxylin staining. This reagent should be replaced immediately.
Hydrate Sections Thoroughly. Slides are thoroughly hydrated prior to hematoxylin staining. The uneven hematoxylin staining visible in the epidermis in this skin section was caused by residual xylene and traces of wax present when the hematoxylin was applied.
Monitor Hematoxylin Quality. Two slides from the same control block are shown. Even macroscopically, the variation in the level of staining is obvious. In this section the epidermal nuclei are poorly defined and are pinkish in color. Avoid Uneven Eosin Staining. This section demonstrates the effect of residual alkali on eosin staining. Monitor Eosin pH. The eosin stain is uniformly very weak and quite unacceptable.
Note that the only components stained with eosin are the red blood cells. With Wright's stain it can be brought out by staining longer and washing less than for the ordinary blood-stain. Those stains which are dissolved in methyl-alcohol combine fixation with the staining process. Top Definitions Quiz Examples regressive staining.
New Word List Word List. Save This Word! A type of staining in which tissues are overstained and excess dye then removed selectively until the desired intensity is obtained.
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