SIP

Subject: histopathology
Topic: special stain

Special stains such as PAS for carbohydrate, Gomori’s Methenamine silver nitrate for fungus and Gordon and sweet’s silver impregnation for reticulum fibres can be performed by an automated staining machine. The automated stainer used in the lab is Ventana NexES.(The procedure had been introduced by Sharon and June’s blog so I am not going to repeat) Some special stains can only be performed manually as the kit for that particular stain is not available or temporally out of stock. Perl’s reaction and von kossa’s stain are example of special stains that can be perform manually.

Perl’s reaction

Function: demonstration of iron and haemosiderin

Principle: Dilute hydrochloric acid is used to release haemoglobin or myoglobin in red cells that is tightly complexed with ferric iron. An insouluble blue compound, ferric ferrrocyanide will be formed once the ferric iron reacts with potassium ferrocynade solution.

Chemical required: 20% aqueous hydrochloric acid
20% aqueous potassium ferrocynanide
Nuclear fast red

Procedure:
1) Dewax, and section to water.
2) Prepare perl’s reagent. (equal part of 20% aqueous hydrochloric acid and 20% aqueous potassium ferrocynanide)
3) Treat with perl’s reagent for 20 minutes.
4) Rinse with distil water.
5) Counterstain with nuclear fast red
6) Wash, dehydrate, clear and mount

Results: iron----dark blue
Nuclear----red

Von Kossa’s stain for calcium

Function: demonstrate calcium deposits.

Principle: tissue on the slide is treated with Silver nitrate solution. Under the presence of ultra-violet light, calcium salt is reduced and replaced by silver deposits, and will be appeared as metallic silver.

Chemical required: 10% aqueous silver nitrate
Nuclear fast red

Procedure:
1) Dewax and section to water
2) Soak slides with 10% aqueous silver nitrate and leave under UV light for 3-4 hours
3) Rinse in water
4) Counterstain with nuclear fast red for 1-3 minutes.
5) Wash, dehydrate, clear and mount in DPX.

Results: calcium----black

Wing Fat
Tg01