MOVAT's pentachrome stain - The world under the microscope

MOVAT's pentachrome stain,

Purpose,

Performing a multiple histological staining with the aim of staining as many tissues as possible in various colors and color variations so that a clear overview of the coupe is obtained.

Introduction,

The MOVAT coloring is a pentachrome (Penta = Greek chemical numeral for 5)[1] staining that was originally developed by Henry Zoltan Movat in 1955. The staining was simplified in 1972 by H.K. Russell[2].

Implementation,

In the German magazine 'Mikroskopie', Jahrgang 2, Nr. 4/2015, Seite 204-208[3], this stain came to the author's attention and decided to give it a try.

The MOVAT staining from the article is unchanged executed with some exception of some staining times (see table).

The material to be stained consisted of young mice of 8 days old. Fixated in Bouin for a period of 6 months. This to decalcify the already formed bone tissue by the present picric acid and acetic acid from the fixative. After fixation the mice were dehydrated starting at 70% ethanol. The mice were not rinsed with water as usual to prevent the connective tissue from swelling. After 70% followed 85%, 95% and 100% ethanol. All steps in 24 hour mode. After the ethanols followed 2 steps xylene. Then the mice were treated in a mixture of xylene and paraplast (50%/50%). Several baths of paraplast followed 100%, after which the mice were molded into appropriate molds (it is important that xylene can no longer be smelled at the last paraplast bath. If this is the case then the paraplast baths should be taken again with clean paraplast and the times should be taken longer.

MOVAT[3]	Recommended	Performed
Alcian blue 20min RT or 10min 56°C	20 min	20 min
Rinse in tapwater	5 min	5 min
Stabilise in ethanolic ammonia	5 min	5 min
Rinse in tapwater	10 min	10 min
Rinse AD (Aqua dest.)	5 sec	5 sec
Core staining Iron Hematoxylin	10 min	5 min
Rinse AD	5 sec	5 sec
Blue in water	15 min	10 min
Brilliant croceine Fuchsine	10 min	10 min
Short rinse in glacial acetic acid 0,5%	≈ 10 sec	≈ 10 sec
Differentiate in phosphorus tungsten acid 5%	20 min	20 min
Rinse in glacial acetic acid 0,5%	2 min	2 min
Ethanol 100%	3 x 5 min	3 x 5 min
Safran du Gatinais	60 min	60 min
Rinse AD	≈ 10 sec	≈ 10 sec
Isopropanol 100% I en II	2 x 4 min	2 x 4 min
Xylol I en II	2 x 4 min	2 x 4 min
Malinol/Euparal/Depex		Depex

Alcianblue[3]:

- Dissolve 1g Alcian blue in 100ml AD (heat to 60 degrees Celsius);

- After cooling, add 1ml of glacial acetic acid 100% and filter.

Iron haematoxylin according to Weigert[3]:

Solution A: Dissolve 1g Haematoxylin in 100ml ethanol 96%;

Solution B: Dissolve 1.16g Iron(III)chroride (FeCL₃ x 6H₂O) in 100ml AD, then add 1ml hydrochloric acid 25%.

Staining solution: Mix solutions A and B (1:1). Staining solution has a shelf life of approximately 1 week. The basic solutions have an unlimited shelf life.

Brillant Croceine-Fuchsine[3]:

Solution A: Dissolve 0.1g Brillant Croceine in 99.5ml AD. Add 0.5ml glacial acetic acid 100%.

Solution B: Dissolve 0.1g Fuchsine acid in 99.5ml AD. Add 0.5ml glacial acetic acid 100%.

Staining solution: Mix 8 parts solution A and 2 parts solution B. Staining solution has a shelf life of several months.

Safran du Gatinais[3]:

- Dissolve 6g Safran du Gatinais in 100ml ethanol 100%.

- For use in an airtight bottle, heat at 50°C for 48 hours and extract the substance[4].

Staining solution has a limited shelf life if airtight. In order to make maximum use of the expensive dye, it is advisable to convert the solution to a usable quantity and thus consume only what is needed.

Results,

The implementation of the staining seems very cumbersome but was not that bad in practice. Of course it is important to prepare a long lasting staining well. Scheduling sufficient time is essential for this. Furthermore, all materials must be ready and put in the right order so that no mistakes are made in the execution. The author uses an excel sheet where all steps are listed and can be ticked off. An example of this is shown on the right.

To make the specimen durable, Depex has been chosen as embedding resin. A statement about the durability of the staining cannot be made.

The specimen pictures shown show the color brilliance of the MOVAT staining.