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Steps to Better Routine Staining + Coverslipping

From patient to pathologist, preparing tissue specimens for histological examination requires care, skill and sound procedures. This guide provides practical advice on best-practice techniques and simple ways to avoid common errors.

Tips for better routine staining + coverslipping are highlighted in this week's guide. We hope each step provides a valuable reminder of good histology practice and also helps with troubleshooting when unacceptable results do occur.

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Step 52 - Use Accurate Timing

 Each step in the staining protocol is accurately timed.

 Step times in staining are approximate and “if we are in a hurry” some steps are skipped. This can produce in consistent results.

These sections were cut from the same block at the same thicknes sand manually stained H&E by different staff members using what was supposed to be the same method.Even macroscopically the inconsistency of the stain can be seen.
Use Accurate Timing

Step 53 - Regularly Monitor Quality

 Control slides are regularly stained to monitor stain quality.

 Control slides are never used for H&E stains. This can make it very difficult to determine whether a staining problem is due to poor reagents, an inappropriate protocol or poor fixation.

This section of kidney includes a variety of eosinophilic tissue elements and well-preserved nuclei that allows a reliable assessment of the quality of H&E staining. Placenta is another specimen type that can be used as a useful control.
Regularly Monitor Quality

Step 54 - Standardize Staining Conditions

 Agitation, wash and drain times are optimized for all steps during staining.

 Agitation, wash and drain times are inconsistent. Solvents and reagents rapidly become contaminated. Staining becomes inconsistent.

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.
Avoid Specimen Trauma

Step 55 - Ensure Complete Dewaxing

 Slide dewaxing is optimized.

 Slide dewaxing is sometimes incomplete and slides contain patches of residual wax. This produces unstained, or unevenly stained areas in sections.

This H&E stained section shows a large unstained area on the left and several smaller areas that are either partially stained or unstained. This is due to incomplete wax removal prior to staining.
Ensure Complete Dewaxing

Step 56 - Renew Reagents Regularly

 Solvents and staining reagents are regularly replaced based on the number of slides stained or racks processed.


 Replacement of solvents and staining reagents is haphazard. They are not replaced until stain quality declines.

This section shows poor quality, muddy hematoxylin staining. This reagent should be replaced immediately.
Renew Reagents Regularly

Step 57 - Hydrate Sections Thoroughly

 Slides are thoroughly hydrated prior to hematoxylin staining.

 Hematoxylin solution rapidly becomes contaminated with alcohol and sometimes xylene. This causes uneven 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.
Hydrate Sections Thoroughly

Step 58 - Monitor Hematoxylin Quality

 The performance of hematoxylin solutions is carefully monitored. During their working life hematoxylin solutions are progressively diluted by carry over from slides and racks and also affected by continuing oxidation.

 Hematoxylin staining is variable from day-to-day and no attempt is made to understand why. For example, the staining bath surface area, the extent of aeration during staining, and the ambient temperature can all affect the oxidation rate.

Two slides from the same control block are shown. They were stained H&E using identical protocols on an automated stainer but with an interval of seven days between runs. Even macroscopically the variation in the level of staining is obvious.
Monitor Hematoxylin Quality

Step 59 - Ensure Complete Nuclear “Blueing”

 Thorough “blueing” of nuclei with Scott’s alkaline tap water substitute or ammonia water is always performed after hematoxylin staining. This requirement is influenced by the natural pH of the local tap water.

 Sometimes nuclei appear pinkish in completed sections due to incomplete “blueing” in alkaline tap water after hematoxylin staining. Nuclei that are under-stained with hematoxylin (or over-differentiated) and over-stained with eosin also appear pink.

A In this section the epidermal nuclei are poorly defined and are pinkish in color. This section was not properly “blued” in alkaline water after hematoxylin staining (skin, H&E).
B This shows another section that was properly “blued” after the nuclear stain. Here the nuclei are much better defined (skin, H&E).
Ensure Complete Nuclear Blueing

Step 60 - Avoid Uneven Eosin Staining

 “Blueing” is followed by a very thorough wash in tap water to remove residual alkali that can impede eosin staining and cause weak and uneven staining.

 Inefficient washing after “blueing” (leaving residualal kali) causes eosin staining to be weak and uneven.

This section demonstrates the effect of residualal kali on eosin staining. Note the patchy nature of the stain (spleen, H&E).
Avoid Uneven Eosin Staining

Step 61 - Monitor Eosin pH

 The pH of the eosin solution is monitored. It is kept close to pH 5.0 to maintain optimal staining. 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).

A section of lung stained H&E. The eosin stain is uniformly very weak and quite unacceptable. Note that the only components stained with eosin are the red blood cells.
Monitor Eosin pH

Step 62 - Thoroughly Dehydrate Before Clearing and Cover slipping

 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.

This section lacks clarity (it appears opaque to the naked eye). Careful examination reveals tiny water droplets to be present throughout.
Thoroughly Dehydrate Before Clearing and Coverslipping

Step 63 - Avoid Drying and Crystal Formation

 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 on the basis of price alone may develop crystals during long-term storage and coverslips may lift.


A This section was allowed to partially dry before cover slipping. This has caused tiny air bubbles to be trapped over some nuclei making them appear black (sometimes referred to as “corn-flaking”).
B A coverslipped section stained H&E showing a multitude of refractile spherocrystals that developed from poor quality mountant within six months of mounting.
Avoid Drying and Crystal Formation

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