[Histonet] RE: morphology on frozen tissues
Dear Sun Zhon,
Your slides should reach room temperature first (either still in a box
or wrapped in plastic) before doing anything. You should prevent them
of getting wet after you take them out of the -80 freezer! We have our
cryo's packed per two sections, face-to-face in Parafilm with little
carton strips between the edges of the two slides. We unwrap them after
the sections have reached room temperature.
I cannot imagine that your peroxide at this low concentration will do
any harm to your sections. The spider web morphology as you describe is
probably due to nuclei that had been damaged by osmotic shock or
something. There can be numerous reasons for this. One of the causes is
for example leaving acetone-fixed cryostat sections too long in
distilled water after staining. You better use tap water instead.
Hope this helps.
Chris van der Loos
Dept. of Cardiovascular Pathology
Academical Medical Center
Amsterdam - The Netherlands
----- Original Message -----
>From Sun Zhon
>Date Wed, 1 Oct 2003 16:44:47 -0700 (PDT)
>Subject [Histonet] morphology on frozen tissues
>These were the steps I processed my frozen tissues, after sectioning,
>air dry tissues for 30 mins fix in acetone for 10 mins at RT air dry
>for 30 mins store in -80 freezer
>The next morning before I did my IHC, I took out the fixed frozen
>tissues from the -80 freezer and put them directly in PBS without
>letting them come to room temperature.
>My tissues looked like spider webs, especially around the edge.
>However, in another experiment I used exactly the same tissues and the
>tissues looked beautiful. The only difference was that while quenching
>the peroxidase, 0.03% H2O2 in PBS was used on the 1st exp (spider web
>morphology); 0.03% in 10% horse serum in 3% BSA was used on the 2nd
>exp (beautiful morphology).
>Do you think the H2O2 will make such a big difference on the
>morphology or there are some other reasons for the spider web effect?
>How do you fix and store your frozen tissues? How do you treat the
>tissues before you do your IHC exps?
>Thank you very much for your help.
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