[Histonet] Re: Decalcified bone eosin overstaining

Thu Oct 2 16:11:55 CDT 2014

What Hazel is saying is true but with acid decalcifiers i.e. formic, nitric
or HCl.  These acids damage nucleic acids aka acid hydrolysisand if the
decalcification is not controlled by endpoint testing, then the nuclei will
appear pale and in some cases, don't stain at all ("over-decalcification").
However, with EDTA is not going to damage the nucleic acids, and you should
have normal hematoxylin and eosin staining.    After EDTA decalcification,
we had good H&E staining.  After acid decalcification, we had over-staining
with eosin and reduced staining time in eosin to 10 continouslu moving dips,
followed by three 95% and three 100% and clearing.  

I think your problem is going from acid/alcohol directly into the eosin, a
no no.   This does NOT allow for correct staining of Eosin at pH 4 to 4.5.
Because you now have such an acidic environment, this is probably the cause
of eosin over staining - often an ugly brick red color.     You should have
running tap water rinsing after hematoxylin for at least 1 minute, also do a
1 minute water rinse after the acid/alcohol.  You obviously answered your
own question "Perhaps a tap water rinse after the Acid Alcohol?" and that is
a resounding YES!!!

Where is your bluing step?  You are leaving your differentiated hematoxylin
in a reddish/blue state.  

A correct sequence for Harris Hematoxylin staining:  

Deparaffinize: 

Xylene x 3 changes

100% x 2 changes

95% x 2 changes

70% x 1 change

Distilled water rinse - 1 minute to hydrate the section

Harris Hematoxylin - up to 10 minutes

Tap water rinse - 1 minute

0.5% Acid/alcohol -  1 dip.  Do this quickly and go right into running tap
water. 

Tap water rinse - 1 minute  Water should flow start under and go up over the
slides.  

Bluing solution - 1 minute  This is a very mild base.  Buy a commercial
solution specified for Harris hematoxylin.    

Tap water rinse - 1 minute

70% alcohol - 1 minute

Eosin - time desired

95% alcohol X 3 changes

100% x 3 changes

Clearing

You will see the sections turn blue after a bluing solution which requires a
tap water rinse and also a 70% alcohol rinse make sure there are no bluing
solution cations carried over into the eosin.   Optimal pH for eosin
staining is 4 to 4.5 (which you have altered by going from acid/alcohol
directly into eosin).    A 70% alcohol also equilibrates to the alcohol
percentage of the eosin solution.   If the eosin still is over staining,
reduce time to 30 sec.  If dipping, be sure you are move the slides up and
down continuously. After the eosin you should be able to just rinse in 95%
alcohols to differentiate out excess eosin.  If any of the post-eosin
alcohol rinses have too much eosin carryover, you will continue to stain the
bone.   Make sure alcohol rinses after eosin are fresh or at least rotated
so there is a fresh last 95% and fresh last 100%.   Any pink color in the
last 100% means you have water carryover which can cause 1) cloudiness 2)
eosin bleeding after cover slipping.    

On the side and many years ago,  we used Harris Hematoxylin for acid
decalcified bone followed by running tap water rinse but eliminated the
acid/alcohol step entirely to avoid even weaker nuclear staining already
caused by acid hydrolysis from the acid decalcifier.  A bluing solution was
used followed by a water rinse before eosin staining sequence.  I learned
this from the AFIP bone lab and it worked with excellent results.   However,
you are working with EDTA decalcified bone and when you eliminated the
acid/alcohol, it worked great without eosin over staining.    

Good Luck

Gayle M. Callis

HTL/HT/MT(ASCP)

You wrote:  

When the bones are over decaled there will be very little hematoxylin
staining and too much eosin.  You can use a buffer before the hematoxylin to
aid the hematoxylin in staining.  I think one/two dip(s) in eosin will be
sufficient.

Hazel Horn

Supervisor of Histology/Autopsy/Transcription

Anatomic Pathology

Arkansas Children's Hospital

1 Children's Way | Slot 820| Little Rock, AR 72202

501.364.4240 direct | 501.364.1241 fax

 <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> hornhv <@t>
archildrens.org

archildrens.org

Sent: Thursday, October 02, 2014 1:11 PM

To:  <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> histonet
<@t> lists.utsouthwestern.edu

Subject: [Histonet] Decalcified Bone Eosin Overstaining

Hey histonetters! I'm currently doing some H&E Staining for EDTA Decalcified
Bone Tissue and it seems that the tissue is staining very intensely with
Eosin...perhaps too much. Was curious to see from some of the more
experienced histotechnicians out there which methods you guys have used in
the past to perhaps fix an issue like this. Here is my protocol that I have
used once I have sectioned the paraffin embedded tissue. I have omitted the
1 second Acid Alcohol Dip in a few of my trials and it seemed that this
method worked great to keep the tissue from overstaining with Eosin.
However, this is an important step for differentiation of the
Hematoxylin...so I'm a little puzzled about what to do next. Perhaps wash
with tap water again after the Acid Alcohol?

1. Xylene - 3 Minutes

2. Xylene 3- Minutes

3. Xylene - 3 Minutes

4. 100% EtOH - 3 Minutes

5. 100% EtOH - 3 Minutes

6. 95% EtOH - 3 Minutes

7. 70 % EtOH - 3 Minutes

8. Filtered Harris Hematoxylin (Commercial Purchase) - 6 Minutes

9. Wash with running Tap water

10. 1 second dip in 0.5% 12M HCL Acid Alcohol

11. Eosin Y Solution - 1 Minute

12. 70% EtOH - 2 Minutes

13. 70% EtOH - 2 Minutes

14. 95% EtOH - 2 Minutes

15. 95% EtOH - 2 Minutes

16. 100% EtOH - 2 Minutes

17. 100% EtOH - 2 Minutes

18. Xylene - 1 Minute

19. Xylene - 1 Minute

20. Xylene - 1 Minute

Trevor Jordan Wait

University of Texas Health Science Center, San Antonio Class of 2017 MD
Candidate Abilene Christian University Class of 2013 Graduate B.S.
Biochemistry _______________________________________________

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