[Histonet] Re: Pam Marcum colleague losing bone sections from slides

Gayle Callis gayle.callis <@t> bresnan.net
Tue Apr 7 16:56:33 CDT 2015


>From Pam:  I am currently trying to stain L6 vertebrae from rabbits. They
have been decalcified and paraffin processed properly. I've tried cutting at
both 5 and 10 microns and my tissue is still not sticking to my slides. I
know my sectioning is fine because I'm successful with every other tissue
I've ever sectioned and stained. For some reason the bone I'm using won't
stick to any slides. I was using charged slides and I even tried
poly-L-lysine slides, but the bone keeps coming up even before I attempted
to stain them. I've even tried leaving them in the incubator for more than
the usual 48-72 hours. I know it's possible to do other stains beside H&E on
bone, but I think my main issue is just getting good contact between the
tissue and slide. If you have any advice or thoughts, I would love to hear
them. 

  

I will get the messages to him ASAP. 

  

Pam 

*************************************************************************

 

What was meant by incubator and at what temperature?   It helps to dry
sections FLAT, at 37 to 40C for several days.  Do NOT dry at 60C.   

 

If the sections are not staying on plus charge or poly L lysine coated
slides,  then use chrome gelatin subbing solution in a water bath OR by
pre-subbing clean microscope slides.    

 

This is the Chrome gelatin protocol that worked for our huge decalcified
bone sections and or problem bone sections. 

 

Chrome Gelatin Subbing Solution:  Section/Slide Coating Adhesive

 

0.1 g Chromium Potassium Sulfate (this is toxic.  Collect for proper
disposal, not down the drain is you pre-sub the slides). 

1.0 g Gelatin:  100 bloom, Sigma.  For large bone sections, use 200 or 300
bloom gelatin, Sigma).   200 and 300 bloom gelatins are very large gelatin
molecules made from pig collagen.  100 bloom is a much smaller molecule than
200 bloom.   Do         NOT use household (cooking)  gelatin used for
cooking. Buy the pure gelatins only. 

1 liter Distilled Water

 

                Dissolve chromium potassium sulfate and gelatin in hot but
not boiling water.  Cool subbing solution before use, and store in
refrigerator.  If gelatin gets growth, discard, make new.  A few crystals of
Thymol in stock subbing solution can help prevent growth. 

 

DO NOT USE PLUS CHARGE SLIDES WITH SUBBING SOLUTION.   GELATIN COATS OVER A
PLUS CHARGE COATING AND NEGATES THE POSITIVE CHARGE.  

 

For presubbing glass slides, wash these by dipping in acetone, air dry
before using the pre-subbing protocol to get rid of any greasy/oily residues
on glass surface.   If you put the subbing solution in a water bath,
uncoated,  glass slides will work fine without further washing.    

 

You can do either of the following: 

 

1.            Add 10 ml subbing solution to a warm water bath for paraffin
sections. Then mount sections onto the cleaned glass slide, drain, and air
dry, store in a cool, dry place. 

2.            Dip acetone washed, dry slides into subbing solution, air dry,
and store in a dust free area.  Box subbed slides and store until needed.  

 

If you get background staining with hematoxylin (hematoxylin stains gelatin)
then dip  pre-subbed slides in NBF ~10 times, rinse with distilled water,
air dry and store slides.  The aldehyde fixative cross links the gelatin to
some degree, but still allows section to adhere without annoying background
staining.  

 

Pick up sections from water bath drain and lay flat to dry at 40C for
several days.   You will not need extra subbing solution in the water bath
if using presubbed slides. 

 

 

IF all else fails, try Sterchi tape transfer method with packaging tape.   I
have the method with photos and publication, and will send privately.   

 

Good luck

 

Gayle Callis

HTL/HT/MT(ASCP)



More information about the Histonet mailing list