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Is this simply freezing artifact? This sample was frozen in an isopentane bath-which had worked well for the previous biopsies.
Brief overview of the working protocol,
-Biopsies frozen in isopentane
-Mounted to cork (OTC on base only)
-Stored in -80C
-Warmed to 20C 1 hour before cryosectioning
-Sectioned at 10um (using anti-roll plate)
-Sections are picked up on room temp slide
-Blocked in 4% NDS for 2 hours (fridge 4C)
-Incubated in the primaries overnight (Room temp (anti laminin and myosin))
-Secondaries for 2 hours (room temp)
Any suggestions are appreciated.
Mon Jul 18, 2011 9:08 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1872
Post subject: Welcome.
Looks to me like a small amount of ice-crystal artefact.
(One can cut 5 freeze say, 5 bxs...4 will be fine but one may have such artefact.
However, main artefact is separation/ dislodgment of muscle fibres and laminin.
Could be due too harsh handling of bx/ not sharp enough blade when cutting/inadequate picking up of section onto slide.
Latter is most likely if all other bxs are fine.
Do you fix before staining?
If not, this can contribute/cause ( I use Hoechst/DAPI to stain nuclei. If you then see "nuclear streaming", this indicates a certain amount of lysis.)
I may be wrong...others' opinions would be welcome.
Nice, strong staining, though
Out of interest....
Why do you incubate in 4% NDS for 2hrs?
Why at 4C?
Why incubate primaries overnight ( at RT)?
Interested.
Carl
Tue Jul 19, 2011 6:23 am
CSAFibertype
Rank: Member
Joined: Jul 18, 2011
Posts: 6
Post subject: Thanks Carl
I am just getting acquainted with the protocol. Our PI chose it and I have been modifying as I go. The stated reason for the NDS is blocking. Temp just as per the protocol. As for the primaries-that is also just following the protocol-however I have experimented with it a bit and found that by increasing the concentrations I can achieve the same results in less time, shortest I have done was 2 hours.
I don't fix before staining-could this cause deformation? I have read that acetone and formalin can interfere with ab binding.
I do use DAPI for nuclear staining, and am seeing quite a bit of "streaming" in this sample. You said the separetion could be due to harsh handling-do you mean at the time of collection before freezing? In which case is the bx a lost cause?
I am consistently getting poor results from this bx-having run it 5 separate times. Is it possible that the water content of the bx is higher than others and thus might necessitate a different cryo-temp?
" I don't fix before staining-could this cause deformation? I have read that acetone and formalin can interfere with ab binding."
Yes...autolysis. Nuclear streaming is indicative of this.
Sure, fixation will "interfere" with Ab binding. It's a balance between stabilising the structure to obtain good morphology and loss of antigenicity.
Proteins like MHC and Laminin are pretty robust and withstand autolysis, as you see from your images of immunostaining of unfixed sections but....other protein will degrade. That's the function of short fixation: stabilisation of structure/Ags.
DNA will unwind if assocciated proteins are not stabilised/fixed: seen as nuclear streaming" with DAPI/Hoechst.
That block should not be a "lost cause"....if you have tried "everything" even tried drying sections for longer, after cutting/mounting on RT slide ( 60 mins under a fan, then immunostaining) then , as a last resort just try defrosting the bx and refreezing it, in exactly the same way.
Keep us posted.
Anyone else have ideas, please??
carl
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Wed Jul 20, 2011 4:05 am
CSAFibertype
Rank: Member
Joined: Jul 18, 2011
Posts: 6
Post subject: Modified protocol
I ran a slightly different protocol yesterday-along side the old one.
Protocol modifications
-Dried the slide for 1 hour after slicing
-Incubated primaries at 4C
It worked great, I will redo the "trouble" bx with the new protocol and post results, should be done tomorrow.
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