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|From:||Michelle Ting <email@example.com>|
|Date:||Wed, 02 Jun 1999 17:09:37 +1000|
WOW! i didn't realise that many people were doing bone immunohistochemistry
and having problems. the advice i received has been fairly specific for the
problem i had with sections falling off my slides, through and after an
antigen retrieval step.
for those with similar problems, here follows some detailed advice that i
received from other histonetters. hope something works for you....
(i also hope that the people who sent the advice don't mind me passing it
on. thanks again to all of them!)
We've been doing ihc on hamster knees for some time and have found that the
only way to keep the sections on the slide was to use gelatin coated slides.
It will give you some background on your slide from the counterstain and the
chromagen. We section the knees let the slides stand upright on a drying
board for one hour, then transfer the board and slides into a 60 C oven for
90minutes. After the oven treatment store your slides on your warming tray
until your ready to use them. It sounds like a lot to go through, but its
worked very well for us. Some other things to keep in mind: the buffer we
were using had detergents, when we switched to a straight PBS buffer we
never lost any sections to lifting or folding. Biogenex has a product that
can be used for bone antigen retrieval that doesn't involve heat. The name
is Decal Retrieval Solution # HK089-5K. It is specifically formulated for
use on acid decalcified tissue. This product has worked very well on our
Try APES coated slides, we very rarely get any floaters no
matter what the treatment........
Fisher Scientific makes a PLUS charged slide that helps the tissues
adhere to the slides and yields no background staining reactions. They
have worked quite well with our immuno's on marine tissues here.
If you can't locate those slides, subbing your slides with Poly-L lysine
before adhering your tissue sections will help also.
Dodi Borsay Horowitz, Research Biologist
We do a considerable amount of bone IHC on formalin fixed formic acid
decalcified specimens. I have a couple of pearls for you.
1) Assure the end-point of decalcification by testing the spent formic acid
with 5 % ammonium oxalate (w/v aq.). 4 parts spent formic : 1 part ammonium
oxalate, presence of white calcium oxalate precipitate indicates the need
for further decalcification. use just enough formic to completely cover the
specimen(s), agitate gently during the day, change the solution daily
checking for end-point (no precipitate)periodically. At end-point,
thoroughly rinse the specimen(s) with running tap water (1 - 4 hours) before
proceeding with processing.
2)Adherent slides. Our preference is to use one of two brands. BD Gold Seal
Ultrastick or Polysciences Sectionlock slides. Use a float bathe temperature
approximately 3-5 degrees below the melt point of the paraffin. No
adhesives, additives or alcohol. Float the sections(we use two sections per
slide spaced widely apart so that one of the sections will serve as an
on-slide no antibody control)until flattened then pick up and drain the
slide vertically 5 - 15 minutes until all the water has drained from the
sections. Then place the slide flat onto a two-stage warmer. The first stage
we set at 5 degrees above the melt point and the second stage we set at 10
degrees above the melt point. Slides spend 5-15 minutes on each stage until
it "looks right". Cool flat overnight. No need for further "baking".
3)Deparaffinize to distilled water as usual. Blot gently and encircle the
sections with a hydrophobic pen or wax pencil (china marker). Place the
slides into a PBS (pH 7.4) solution containing 0.02 % Triton X-100
(Aldrich/Sigma). Heat the antigen retrieval buffer in a microwave oven to
near boiling (95 oC) remove the solution from the oven and then place the
slides into the hot solution. Let the slides set in the hot solution on the
counter undisturbed 15 minutes. Rinse the slides in two changes of
PBS-Triton 3 minutes each.Then proceed.
Hope this helps, Good luck, Donna Montague, UAMS Orthopaedic Research,
Little Rock, AR, USA
Have you looked into using something other than Formalin for fixation
so you do not have to do antigen retrieval? We used to do IHC on boney
tissues that were formalin fixed and formic acid decalcified. We would
have to do antigen retrieval and the tissues would usually fall off.
We then switched to using Zinc Formalin which prevented the need to do
antigen retrieval. Also, we have our sections cut onto Plus slides,
which are charged and keeps the tissue on better. I've been really
pleased with the results.
I cut the sections at 6un and put on 2% ATS coated slides. The slides were
put on their SIDE in a tray on a hotplate at 45C for half and hour and then
into a 37c oven overnight. Antigen retrieval was done in the microwave 70%
power, bring up to 55C, stop and leave for 5 minutes. No problems.
Since, I have been doing Serology sections on sheep teeth. Was having
problems,with sections lifting off, tried the above, still problems. So I
rang the "mother of histology" here in Dunedin and she told me to leave the
sections at Room temp on their side in a tray for at less half and hour and
then into the 37C oven for an hour( but I decided to leave them overnight),
then place into 60C for half and hour before staining. What a difference.
Let me know how you get on
School of Physiology & Pharmacology
The Universitiy of New South Wales
ph 9385-3810 fax 9386-1059
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