There are many stains out there to histologically stain for calcium but we use primarily 2. They both give different color results and use different chemicals. The first is called Alizarin Red S. This stain can be visualized with or without a polarizing microscope. The polarized microscope makes the calcium deposits much brighter (see picture below). As the picture suggests, we also carry positive control slides for this and other calcium stains. We like this one the best because it is a much quicker stain that can be completed in 25 minutes using paraffin slides. The chemicals necessary for the stain are alizarin Red S powder and ammonium hydroxide. You also need a pH meter to get the solution to a pH range of 4.1-4.3.
The other stain is Von Kossa‘s method for calcium. This is a much longer stain (1 hour 41 minutes) that uses silver nitrate to turn the calcium salts black. To achieve good results with this stain, the tissue sections must be exposed to direct sunlight, a UV lamp or a 100 watt lamp light while immersed in the silver nitrate. In our opinion this stain is more finicky and easier to foul up than the Alizarin Red stain. Unlike most silver protocols, the glassware in this protocol will not turn black. The Von Kossa solution protocol will be posted on the protocol solutions the day after today.
After the stain is complete and the controls tissues have been verified, the toning solution (gold chloride) can be used to clean all glassware. Keep in mind that this solution is very toxic and should be disposed of properly (not down the sink).
I just found this amazing resource for scientific experiments and more. They have over 2500 videos that teach laboratory fundamentals. Their video categories include; general, neuroscience, immunology and infection, clinical and translational medicine, bioengineering, applied physics, chemistry, behavior, and environment. I just watched DNA extraction from paraffin embedded material for Genetic and Epigenetic Analyses. I can now go try this in my lab. Wonderful, I hope this helps!
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.
Here are the other 2 pictures that were going to be in the previous post, plus 6 more. The other 6 are also 10x,20x, and 40x of 2 different tissues. I can not imagine what life would be like without flexibility.