More on [Histonet] Looking for detailed protocol to stain for PASin liver sections (paraffin)

Wed May 7 18:17:57 CDT 2008

I have found the following Modified PAS procedure especially useful for
cytology smears that have been fixed in ethanol:

Alcoholic PAS Stain

Mucins and glycogen are water soluble. Over-rinsing slides, especially
cytological smears, could result in excessive loss of these PAS
substances. The following variant of the PAS stain performs the
reactions in alcohol, thus decreasing the loss of these substances.

Solutions:

1.	Alcoholic Schiff reagent 

     Basic Fuchsin 0.5g 
     Ethanol 80ml 
     Distilled Water 20ml 
     Hydrochloric acid 1ml 

2.	Alcoholic Periodic acid
Periodic acid 	0.5g
95% ethanol	50ml

Procedure:

1.	Fix smears in 95% ethanol or use methanol fixed air-dried
smears.
2.	Place in alcoholic periodic acid 20min.
3.	Wash in alcohol
4.	Place in alcoholic Schiff's 20min.
5.	Rinse slides in alcohol to remove excess dye.
6.	Rinse slides in water.
7.	Counterstain slides in Haematoxylin 2min.
8.	Rinse slides in water.
9.	Differentiate and Blue.
10.	Dehydrate, clear and mount.

References:

Horobin and Kevill-Davis (1971) Stain Techn 46:53-58.

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Gayle
Callis
Sent: Thursday, 8 May 2008 2:06 AM
To: Histonet
Subject: Fw: More on [Histonet] Looking for detailed protocol to stain
for PASin liver sections (paraffin)

Dr. Nasonkin was staining for glycogen (per my question) and via private

emailing, it was suggested he not use aqueous formalin or PFA fixation,
but 
an alcoholic fixative to retain the glycogen in the liver, do diastase 
digestion to prove it is glyogen, and also process tissues -  starting
in 
higher (100%) alcohols to help retain glycogen.   Suggested fixatives
were 
Carnoy, Gendre and alcoholic formalin.

If you have any other suggestions for him, please add to the list of to
do's 
.

Gayle M. Callis
HTL/HT/MT(ASCP)
Bozeman MT

> ----- Original Message -----
> From: "Igor Nasonkin" <nasonkini <@t> mail.nih.gov>
> To: "Gayle Callis" <gayle.callis <@t> bresnan.net>
> Cc: <mpotok <@t> umich.edu>
> Sent: Tuesday, May 06, 2008 6:35 PM
> Subject: Re: [Histonet] Looking for detailed protocol to stain for PAS

> in liver sections (paraffin)
>
>
> Gayle,
> Yes, glycogen.
> Does it mean that if we fixed livers in paraformaldehyde prepared on 
> PBS
> we
> lost glycogen? What if we fixed the whole liver, not section on a
slide? 
> One
> cannot remove glycogen from the whole liver this way. Thank you for
this
> info,
> Igor
>
> Dr. Igor O. Nasonkin
> Research Fellow
> National Institutes of Health/NEI
> 9000 Rockville Pike, MSC 1864
> Bldg 10, Room 10B11
> Bethesda, MD 20892
> Tel: 301-443-7398 -work
>       617-388-4104 -cell
> Fax 301-480-1769
> email: nasonkini <@t> mail.nih.gov 
> http://www.nei.nih.gov/intramural/nnrl.asp
>
> On 5/6/08 8:08 PM, "Gayle Callis" <gayle.callis <@t> bresnan.net> wrote:
>
>> You did not say what you are trying to see in the liver?  Glycogen?
Some
>> other PAS positive tissue component?   If so,aqueous formalin
fixation
>> will remove the glyocgen.  An alcoholic fixative helps retain 
>> glycogen.
>>
>> Gayle M. Callis
>> HTL/HT/MT(ASCP)
>> Bozeman MT 59715
>>
>> ----- Original Message -----
>> From: "Igor Nasonkin" <nasonkini <@t> mail.nih.gov>
>> To: <Histonet <@t> lists.utsouthwestern.edu>
>> Sent: Tuesday, May 06, 2008 5:24 PM
>> Subject: [Histonet] Looking for detailed protocol to stain for PAS in

>> liver sections (paraffin)
>>
>>> I am looking for a detailed protocol for PAS staining in liver 
>>> sections (paraffin-embedded). The 1st set of sections did not work, 
>>> and we had no
>>> +control since we have not done it. These are 4-6wk mouse liver 
>>> +sections
>>> embedded in paraffin; our lab is experienced in IHC on paraffin 
>>> sections so these were done right. But PAS staining did not work. 
>>> What could be main reasons? Any positive control we could use? Thank

>>> you in advance, Igor

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