[Histonet] Re: Schiff's storage temperature - long reply

[gayle callis]

Dear Histonetters, 

 

At the risk this may be more information than you need.   My fascination
with PAS and Schiffs reagents prompted the following.  

 

Our Schiffs reagent,  prior to available commercial products,  was made in
house.  We made Lillies Cold Schiffs solution stored at 4C according to
protocol but no time limit was given.    Another in house Barger and
DeLamater reagent said several months at 4C - a rather vague time.  Luna
said 3 months storage at 4C.     I think the kicker here is to test any
Schiffs before use since it is unstable over time to remove any doubt.
Other differences is  basic fuchsin versus pararosaniline dyes and there are
publications discussing these dyes for Schiffs reagents.   Confusing - yes!!


 

Back in the dark ages when practical exams were still done for HTL,
curiosity led to testing both in house and a commercial Schiffs reagent.
All staining was done at RT in parallel fashion using fresh in house and
commercial Schiffs on adjacent sections/separate slides.   We noted no
visible staining difference i.e. intensity.    This led exclusive use of
commercial Schiffs to avoid last minute, tedious and exposure to
carcinogenic basic fuchsin i.e. the weighing out dry,  and strong HCl fumes.
We didn't always have good fume hoods back in the dark ages of histology.
Old habits die hard to store Schiffs at 4C, so RT storage was ignored, and
4C prevailed with commercial Schiffs with staining at RT.     

 

However, if I had to have immediate PAS staining in a busty lab, I would
store Schiffs at RT to avoid waiting for equilibration to RT.   This brings
up a question.  Has the person asking at temperature storage tried staining
with cold versus warm as the staining times I saw i.e. up to 30 minutes
could very well make up the difference.  Cold Schiffs longer time versus
warm Schiffs shorter time?   1% versus 0.5% periodic acid will also make a
difference.   

 

The best discussion of Periodic acid Schiffs reaction staining mechanisms
and technical comments can be found in Sheehan and Hrapchak Theory and
Practice of Histotechnology, Carbohydrate Chapter 9, p9 164 166.  Second
Edition, 1980.   This book is a classic and still available in Paperback
form from NSH.     This gives excellent hints on performing the stain i.e.
1) always used freshly prepared periodic acid (PA) something Culling
insisted on.  PA is cheap and goes into solution immediately.   I do not use
premade periodic acid solutions in kits as I don't consider these freshly
made, simply unused but made days, weeks, months before purchasing the kit.
If they are used, should be discarded and go to making PA fresh daily.
Another finding was a decision to preweigh  dry aliquouts of  PA into a
clear glass Erlenmeyer flasks to save time, seal the top of flask tightly,
store on shelf until distilled water was added.  This did NOT work as  the
preweighed PA went bad due to exposure to air, and probably light.  PAS
staining was poor, renal pathologist complained, and was embarrassing.
Freshly weighed, solution prep daily was reinstated.          

 

Some things we did to maintain good PAS staining. 

 

1.  Since shelf life varies with use and probably storage conditions,
expiration date is important but in house Schiffs could go bad before that.
This is probably true of commercial solutions too.   

1.  If Schiffs reagent turns pink, discard.  

3.  Never freeze Schiffs reagent.   Shipment to cold weather states i.e.
Montana in winter was avoided after Schiffs arrived frozen.   Plan ahead.   

4.  Never return used Schiffs to stock bottle.   Store in another bottle,
dated and tightly sealed.  Expendable plastic wrap makes a good tight seal
around and under lids on bottles or Coplin jars with used Schiffs.     

6. Test used and even new if the latter hasn't been used for a time after
opening stock bottle.  Sheehan and Hrapchak have test in chapter.  We just
used a few drops of Schiffs in NBF and watch the bad color.......deep
blue-purple.   You want instant bright red with slight tinge of purple. If
the lab is formalin free, use histologic test by staining cross section of
human appendix, look for good staining of fine meshwork sarcolemma between
smooth muscle cells. 

7.  Reuse Schiffs for up to a week, unless it turns pink.  

8.  Do not dump Schiffs down the drain, discard using appropriate chemical
safety facility.

9.  Don't get Schiffs on your skin.......nitrile gloves.   Even though some
vendors may have Schiffs removal reagents, once on the skin, you are already
exposed to basic fuchsin - the contamination/exposure horse is already out
of the barn. 

10.  Sulfurous acid rinses were discontinued after hearing Culling (in
person) lecture on PAS staining.  He said a 10 minute running tap water
rinse was sufficient to intensify the color and we never had a problem using
only water rinse.  

 

We have used Sigma's Schiffs, and also Fisher reagent grade Schiffs with
great success.   I think commercial solutions are wonderful, and you may
find subtle differences in a given lab's storage, usage and conditions.  If
they work, then use them but be aware of limitations.  If RT storage speeded
up my time for results, then I would do just that.   Otherwise, I would
still store a 4C, equilibrate to RT.  Remember there are people who use
microwave heating for a PAS stain.   

 

PAS success also depends on what structures are being stained and thickness
of sections.  Our 2 µm renal biopsy section protocol had different timing
that staining 5 µm sections from other organs.   

 

If anyone wants this chapter, I will be happy to scan to pdf and send via
private email.  

 

Hope your weekend is a good one

 

Gayle M. Callis

HTL/HT/MT(ASCP)