Bone forming in various histological stains - The world under the microscope

Bone formation or ossification[2] visualized by various histological stains.

To observe the staining of bone forming tissue and the differences in staining, it was chosen to cut sections of similar tissue which was fixed in the same way. For this purpose, five-day-old mice were fixed in Bouin liquid. To allow the fixative to penetrate the tissue more easily, the extremities were removed and small incisions were made in the skin. Tissue was left in Bouin for approximately two months after which dehydration was started in ethanol 70%. The usual rinsing out of the fixative in water did not take place to prevent tissue from swelling too much[3]. Further ethanol steps were 85%, 95% and 100% with a duration of 48 hours per step. Via xylene, the switch to paraplast plus was then made. To ensure that all xylene was replaced with paraplast, three liquid paraplast baths were used.

Tissue was then cut on an A&O 820 rotary microtome with Leica 818 high profile microtome blades. After drying, the paraplast was removed via 2 xylene baths and the sections hydrated in an ethanol series of 100%, 95%, 85%, 70% and 50%. Staining which was performed were:

- Haematoxylin/Eosin[8];

- PTAH (Phosphorus tungsten haematoxylin)[7];

- Van Gieson[9];

- Trichrom according to Mallory from 1900[5];

- AZAN according to Heidenhain[6];

- According to Masson-Goldner[4].

Dyeing protocols are mentioned below and are taken from 'Romeis Mikroskopische Technik' see Source Notes. The preparation of the dyes was carried out as described in Romeis but the staining times were adjusted empirically[2] for best results. Also dehydration after staining was often performed with isopropanol instead of ethanol 100% due to the high cost of absolute alcohol. After drying of the resin, images of the sections were taken with a Moticam 2300 digital microscope camera mounted on a Leitz Orthoplan microscope.

Source reference: