The Staining :)

Hi everyone, it is my turn to post for this week. For this month, I have been posted to the routine staining area; thus, I have been exposed to various special stains like CAB, PAS, PASD, MT, Orcein, Fe, Ziehl Neelsen etc.
Today, I will introduce on PASD and Fe stain.

PASD (Periodic acid Schiff diastase)

Function: It helps to differentiate glycogen from other carbohydrates

Principle: Glycogen is digested by diastase where no reaction will occur with Schiff’s reaction afterwards.

Procedure:
1) Dewax and bring section to water
2) Treat section with diastase
3) Wash well with water
4) Treat with 1% aqueous periodic acid for 5 mins
5) Wash in water
6) Treat with schiff’s solution for 5 mins
7) Wash in water
8) Counter stain nuclei with haematoxylin
9) Blue in water
10)Dehydrate, clear and mount in DPX

Result:
Any presence of glycogen will be stain magenta on the PAS stained slide while for the PAS/D stained slide, there will be absence of it.

Note:
A positive control must always be used




A picture showing presence of glycogen stained magenta (PAS/D stain is used). This suggests that the patient is suffering from glycogen storage disease



Fe (Perl's reaction)

Function: Demonstration of iron and haemosiderin

Principle: Dilute hydrochloric acid solution release ferric iron that is tightly complxed to protein as hemoglobin. The ferric iron will then reacts with potassium ferrocyanide solution to produce an insoluble blue compound which is ferric ferrocyanide.


Reagent preparation:

Perl's reagent
20% hydrochloric acid -- 5 ml
20% potassium ferrocyanide --5ml


Procedure:

1) Dewax and bring section to water
2) Treat with perl's reagent for 20minutes
3) Rinse in SRW
4) Counterstain with nuclear fast red for 3 mins
5) Wash, dehydrate, clear and mount in DPX

Result:

Iron -- dark blue
Nuclei -- red




Perls Prussian blue stain showing hemachromatosis in cardiac muscle

Control: A positive control must also be used in this case


Staining is a very challenging job whereby you need to be able to multitask, however, i learn a lot of many different stains and the different things that need to be observed under the microscope.

Feel free to clarify any doubts and i will try my best to answer them :)

References:

http://images.google.com.sg/imgres?imgurl=http://img.medscape.com/pi/emed/ckb/pediatrics_genetics/941088-941089-941632-941708.jpg&imgrefurl=http://emedicine.medscape.com/article/941632-media&usg=__r1ZzgRvxBDwxsQKDdIiuE03bghw=&h=346&w=573&sz=37&hl=en&start=2&tbnid=x8c9fQVuwvM8JM:&tbnh=81&tbnw=134&prev=/images%3Fq%3Dperiodic%2Bacid%2Bschiff%2Bglycogen%26gbv%3D2%26hl%3Den

http://images.google.com.sg/imgres?imgurl=http://www.gladstone.ucsf.edu/gladstone/files/histology/Perls-Prussian-blue-stain.gif&imgrefurl=http://www.gladstone.ucsf.edu/gladstone/site/histology/section/2288&usg=__GqcH8PvwJO0Uffc2OAIeVfzDNgU=&h=331&w=504&sz=44&hl=en&start=7&tbnid=RpRio2w7otbr2M:&tbnh=85&tbnw=130&prev=/images%3Fq%3Dperls%2Breaction%26gbv%3D2%26hl%3Den%26sa%3DX




Cheers,
Zi Shuang
0703383J