We need to ensure quality of H&E sections, so when reviewing the daily H&E, we are looking for blue nuclei with chromatin and a well stained nuclear membrane. Also we are looking for 3 shades of eosin, light, medium and dark red/pink. Some of this is determined by the kind of eosin used. If a control is questionable we should run a second control that we are sure of to ensure that the problem, is a staining problem and not a tissue problem. Over decalcified, over heated or poorly processed/fixed tissue can stain poorly even if all solutions in the H&E stain are OK. When doing H&E on frozen sections extra care must be taken if the slides are fixed in Formalin after cutting. Formalin or concentrated formaldehyde can act as a reducing agent. The best way to remedy this problem is to change the hematoxylin frequently. Frequently can be defined by the lab. Some labs like clinical hospitals, where the volume is high, may determine a frequency based on number of slides, day or weeks. Clinical labs are usually controlled by the pathologist, therefore whatever the pathologist wants, is what will become the norm. For smaller labs this is not the case. Generally a smaller lab will inspect the color intensity of the hematoxylin on a weekly basis to determine the right time to change it. As a rule of thumb, hematoxylin must be filtered before each days use. This helps to keep the floating precipitate out, removes any extraneous cells and lets it keep longer. The time in the 95% alcohols after eosin is where the three shades of eosin are differentiated so these times may need to be adjusted for best results.
A reducing agent (also called a reductant or reducer) is the element or compound in an oxidation-reduction reaction that donates an electron to another species. Because the reducing agent is losing electrons, we say it has been oxidized. (http://en.wikipedia.org/wiki/Reducing_agent)