Dears Jacqui and Ray,
I just want to thank you for all your thoughtful insights and the great
ideas regarding my quest within the IHC world. I will let you know what
happened and hopefully you can read my published results later (rather
sooner than later). I just have to agree with Ray regarding the need for
collaboration between these 2 areas that seem (or we make them look
like) to be very different.
From: firstname.lastname@example.org [mailto:email@example.com]
Sent: Thursday, February 07, 2008 10:47 AM
To: Jacqui Detmar; Leyva-Grado, Victor;
Subject: RE: [Histonet] IL1 KO mouse and cytokines in neurons
Jacqui's suggestion seems super. Localizing something that is not being
(or rarely) secreted seems logically to have less associated problems
than localizing something that by its very nature is mobile and you are
trying to discriminate it from a cell right next to it. Congratulations
on your KO/IHC luck. Wish I had had some of that luck in a former life.
Shows the need for collaboration and understanding between IHC types and
molecular research types.
"This is a -/- knockout to (x)" is insufficient communication to be
assured that (x) won't be staining and costing weeks of frustration and
excess work and needless work.
-------------- Original message ----------------------
From: "Jacqui Detmar"
> Hey there. Yup, I agree that using KO mice as controls can be a
> problem, although I work with about a dozen different KOs and have had
> no problems, so I think it's a matter of knowing *what exactly* has
> been knocked out, plus keeping your fingers crossed and praying to the
> right gods . Anyway, if you're worried about cytokine signaling
> to adjacent cells (which was great thinking, Ray!), perhaps another
> approach is to use either caspase-1 KO (mice available commercially)
> caspase-11 KO mice. These enzymes process pro-IL-1alpha and beta to
> their active, secreted forms. Thus, the pro forms should be retained
> within the primary cell types synthesizing the cytokines. Both
> caspase-11 and caspase-1 KO mice have been reported to exhibit very
> low levels of secreted, processed IL-1beta and IL-1alpha. I have not
> yet seen anyone produce double caspase-1/caspase-11 KOs.
> Jacqui Detmar, Post-doctoral Fellow
> Samuel Lunenfeld Research Institute, room 876
> Mount Sinai Hospital
> 600 University Avenue
> Toronto, ON, Canada
> M5G 1X5
> phone: 416-586-4800 x2451/x2290
> fax: 416-586-8588
> email: firstname.lastname@example.org
> From: email@example.com [mailto:firstname.lastname@example.org]
> Sent: Monday, February 04, 2008 5:39 PM
> To: Jacqui Detmar; Leyva-Grado, Victor;
> Subject: RE: [Histonet] IL1 KO mouse and cytokines in neurons
> Victor and Jacqui,
> Could "some" neurons appearing to be immunoreactive to TNF alpha or
> Il-1 beta simply be the fact that they are cytokines? Small molecular
> weight, they are meant to be released from cells and not membrane
> bound there or as part of the cell structure. Glial cells, by the
> very fact of what they are, are intimately associated with neurons.
> How was tissue fixed? Maybe they are dispersed or leaking to a
> neighboring neuron. Many targets are anchored where they are, such as
> your NeuN label, and that target is not going anywhere. Cytokines do.
> The references of 10-15 years ago were novel then. Not so now. TNK
> K/o as you said you can get. At Wash State, you should have a good
> mouse transgenics and mouse k/o husbandry facility. With some
> Balb/c's, they should be able to generate IL-1 alpha -/-. beta -/- or
> alpha/beta double knock/outs. Not that difficult for a good knock out
> genetics lab. If you are using the&n bsp;knock outs as control for
> the IHC staining (and this applies to any use of knock-outs as
> negative controls for any IHC
> staining) be sure you know EXACTLY what is knocked out. A mouse
> frizzle/frazzle knock-out does not imply the total absence of mouse
> frizzle/frazzel gene and protein. One particular coding exon in a
> multi-exon gene could leave the protein unresponsive or unusable
> in-vivo or shortened but enough might be left, and if the proper
> epitope is left, for IHC staining to occur. Knock out is not the same
> as complete absence. So you can stain something that is "knocked out"
> and that is why you need to be aware of EXACTLY what is knocked out.
> Many people, including yours truely, has been caught like this.
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