Mid-saggital coupe of a mouse - The world under the microscope

Mice are bred and removed from the nest six days after birth. Anesthesia has been performed with Ether. Extremities were removed with surgical scissors and on both sides of the trunk the skin was opened with two incisions to facilitate the penetration of the various fluids. The tissue was then fixed (immersion fixation) with the Bouin fixative[1] in the following proportions:

- 15ml picric acid 1.2%;

- 5ml formalin 40%;

- 1ml glacial acetic acid 98%.

The tissue has been in the fixative for several weeks before dehydration started. In the fixative according to Bouin, this has no harmful effect on the tissue[4], which is the case with many other fixatives such as: Susa, Carnoy, Zenker et cetera. After the tissue has been carefully fixed, dehydration is started. After Bouin fixation it is usual to start directly in Ethanol 70% without rinsing in water. Experience has shown that this way the connective tissue swells less than when first rinsed out in water.

The ethanol range used is: 70%, 85%, 96% and 100% isopropanol (2-propanol). The isopropanol can of course be replaced by ethanol 100%. Usually cost considerations are a good reason to choose isopropanol and it has been proven that isopropanol is excellent for a subsequent paraffin embedding. However, one should take into account that isopropanol penetrates more slowly than ethanol. The selected times in the ethanol range is 24 hours. However, the isopropanol step is 48 hours and the isopropanol has been refreshed halfway through to ensure that all water has been removed from the tissue. After the isopropanol 100% the tissue remains in xylol for 48 hours. Then 72 hours in a heated mixture (1:1) of xylol and paraplast. Here the xylol is slowly expelled by paraffin. This step is followed by three steps of pure paraplast. The reason for this is to ensure that all xylol is replaced by paraplast. A paraffin block that is not completely free of xylol will cause a lot of problems on the microtome and will not produce good cuts. It is important that the temperature of liquid paraplast does not exceed 62°C because otherwise the additives, which are incorporated in paraplast to improve the penetrability and cuttability, will start to decompose. The tissue must remain in the liquid paraffin for a fairly long time. It has been chosen for 72 hours in each bath. After the tissue has been processed into paraffin blocks, cuts of 4μm are cut on an A&O 820 rotary atom fitted with a Leica high profile 818 disposable knife. Hot water stretched slides were mounted on slides from the company Menzel with a standard size of 26 x 76 mm (ISO Standard 8037/I). After the paraffin was removed in a xylol bath and an aqueous state was reached by a descending ethanol series, various stainings were carried out. After staining, the coupes were permanently covered with Euparal. After drying, images were taken on a Leitz Orthoplan microscope equipped with a Moticam 2300 digital camera.

AZAN according Heidenhain[2],

[1]  Prof. Dr. Peter Böck (2010, 18., Edition), Romeis Mikroskopische Technik, ISBN 13: 9783827416766,  München. Verleger: Spektrum Akademischer verlag. Chapter 2,  "Präparationsmethoden", Tabel 2.10, page 90