I agree with Patsy about cleaved caspace 3 staining just apoptotic cells. In a well regulated and defined tissue analysis, TUNEL can certainly be very useful but if you are in an unknown system and tissue milieu where necrosis might, or might not be happening, cleaved caspace-3 is better and discriminates apoptosis from necrosis but there are instances, again in defined systems, where TUNEL is a very, very useful and well established procedure. The comment about how few cells stain I think is right on. Programmed cell death is meant absolutely to proceed from death hit to completion very, very quickly. Cells don't "apoptose" for day's. So when you fix the cell, you are catching it at one particular moment in time and if not in rapid apoptosis at that moment, you miss it. In grad school I worked in mouse and primate thymus where 90%?, 95%?, 98%? of thymocytes undergo apoptosis during negative selection. Did immunoEM and was constantly amazed at how VERY FEW T cells looked so
good and so VERY FEW were in the classical morphologic throws of apoptosis. Once they get "the hit" their brief journey to apoptotic debris is amazingly quick. You are just catching some during fixation that are undergoing morphological apoptosis even is most are destined to do so.
-------------- Original message ----------------------
From: "patsy ruegg"
> In my experience CC3 does a much better job of staining just apoptotic cells
> and not necrotic cells than Tunnel as a matter of fact I have never doubted
> that the staining I get with CC3 is anything but apoptotic cells which is
> not the case with Tunnel, we could not distinguish necrosis from apoptosis
> with Tunnel.
> One complaint I get from my clients is how few cells stain with CC3, but I
> think it is real and they are used to seeing more cells stain with Tunnel
> because Tunnel is picking up necrotic cells as well as apoptotic cells.
> Patsy Ruegg, HT(ASCP)QIHC
> 12635 Montview Blvd. #216
> Aurora, CO 80010
> fax 720-859-4110
> -----Original Message-----
> From: firstname.lastname@example.org
> [mailto:email@example.com] On Behalf Of GT Hebert
> Sent: Thursday, March 27, 2008 8:03 AM
> To: firstname.lastname@example.org
> Subject: [Histonet] Caspase-3 Antibody; other Abs -- Apoptosis vs. Necrosis
> Hello all,
> Does anyone know if Caspase-3 antibody is suitable for detecting apoptotic
> cells (as opposed to both apoptotic and necrotic cells)?
> If it cannot distiguish between cells types, is there an antibody (for
> mouse tissue) that can be used or is my only option in situ techniques?
> Thank you.
> Gustave Hebert
> Scientist II
> Wyeth Research
> Cambridge MA
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