7. Hydrating
In order to stain the preparation, all paraffin must first be removed. The sample is transported in a descending alcohol series to an aqueous environment. To use liquids sparingly it is practical to use small plastic or glass jars. Stick a sticker on them to make sure what is inside. Because the object glass will stand upright in the liquid, it is advisable to place the coupes at the end of the object glass. The descending range consists of 2x xylene, 100% isopropanol, 96% ethanol, 85% ethanol, 70% ethanol, 50% ethanol, demineralised water (the 50% step is allowed but not strictly necessary). The samples remain in each bath for 4 minutes. To ensure proper penetration of each substance, the slides must be moved frequently in the liquid. When the xylene is cold the paraffin dissolves poorly. Warming up to about 25°C ensures a better removal of the paraffin. After about 50 slides the xylene must be refreshed as it becomes saturated, this also applies to the ethanol and isopropanol steps. When changing baths, bring as little old liquid as possible with you (allow to drain at the edge and press briefly on a tissue). After the specimens have arrived in water they may remain in this for a longer time (a few hours is not a problem). Now they can be stained. The images below show the series.
8. Staining
Staining the coupes is the most difficult part of histology. Microtome cutting wasn't easy, now the masterpiece begins. I sometimes compare it to frying a steak. Basically anyone can do that, but only a master chef gets it tasty and juicy. So is staining. In the book 'Mikroskopische Technik' from Romeis many recipes can be found. This book is a must for every histologist. The green copy shows the older edition, 17th edition, which in my opinion is the best. The newest 18th edition¹, red copy, often goes too far for the amateur. It usually requires instruments and dyes that are very expensive and dyeing methods that are too difficult. Think of immunohistochemical dyeing.
It goes too far to name all the details of staining here, the staining technique is too complicated for that. However, it is worth mentioning:
- Progressive staining is a technique in which the coupes stay in the dye for as long as they are stained enough.
- Regressive dyeing is a technique in which first overstain is applied and then the correct level of stain is achieved by means of differentiation (washing out with a suitable liquid).
- One can stain a coupe with a single dye, but double, triple and multiple dyeing are also possible.
- The staining depends on, among other things: staining time concentration of the staining solution, temperature, compartment thickness, fixation.
Four pictures of different stainings.
Fig 1, Tooth aptly stained with PTAH. (PTAH=Phosphorus tungsten haematoxylin)
Fig 2, Same tooth in a Trichrome coloring according to Mallory.
Fig 3, Spine in an AZAN coloring according to Heidenhain.
Fig 4, Jacobson's organ of a mouse in the classic HE coloring.
The HE (Haematoxylin/Eosine) staining is a routine staining in Histological and Pathological Laboratories.
Dyes must be purchased. Ready-to-use dye solutions can be purchased from e.g. Chroma in Münster (Germany). The disadvantage of this is that the solutions lose their tinting strength with the passage of time. A better method is to purchase dyes in solid form. The Aniline Blue/Orange G is a ready-to-use mixture, the acid Fuchsine however is a staining powder. How it should be processed to a staining solution is described in 'Romeis'.
