Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/autofluorescence-in-liver-tissue-tp7580412p7580423.html
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Hi Doug,
Tyramide signal amplification, with 11 washes after the primary, 11
washes after the 2ndAb-HRP (need to re-titer the antibodies compared to
other methods ... and keep the tissue submerged at all times - my thanks
to Richard Burry for the tip - see his Immunocytochemistry book). For
killing the HRP between rounds (if multicolor TSA is needed) see
peroxAbolish info in
Quantitative in situ analysis of FoxP3+ T regulatory cells on transplant
tissue using laser scanning cytometry.
<
http://www.ncbi.nlm.nih.gov/pubmed/21929847>
*Takahashi* H, Ruiz P, Ricordi C, Delacruz V, Miki A, Mita A, Misawa R,
Barker S, Burke GW, Tzakis AG, *Ichii* H.
Cell Transplant. 2012;21(1):113-25. doi: 10.3727/096368911X586747. Epub
2011 Sep 16.
PMID:
21929847
You are not required to use the kit (Mol Probes or PerkinElmer sell TSA
kits) HRP - or other components (except the fluorescent tyramide of
course). I still have not tried it (not needed so far), but am intrigued
by the HRP80 at
http://www.fitzgerald-fii.com/streptavidin-poly-hrp80-conjugate-65r-s118.html
If anyone has experience - either good or bad - with HRP80 or HRP50 or
HRP20 - please let us know here on the listserv or tell me privately.
George
p.s. with respect to HRP80 * TSA ... positive specimens might need a
warning: "do not look through the eyepiece with remaining eye" (yes,
that's a joke - eyesight should recover after a few minutes). If you
don't do enough washes, everything will be blindingly bright. Also,
liver may have endogenous biotin sticking out, so need to make sure to
block well (and always do the controls) when using streptavidin
conjugates like HRP80.
On 6/4/2013 12:23 PM, Cromey, Douglas W - (dcromey) wrote:
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> I am working with a lab looking at IF stained sections of formalin-fixed, paraffin embedded pieces of mouse or rat liver. If there was any actual staining in their first try on our confocal, it was largely washed out by the lovely and strong autofluorescence. Because their animal experiment takes 8 weeks, it's not trivial (or inexpensive) to get new tissue, so they have been using existing blocks. No one in their lab had the foresight to put up snap-frozen tissues for cryosectioning. I have them looking at this document:
http://www.uhnres.utoronto.ca/facilities/wcif/PDF/Autofluorescence.pdf but I'm wondering if liver is always a problem with autofluorescence, or is there maybe something in this lab's sample prep protocol that's making it worse. Any ideas?
>
> Doug
>
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> Douglas W. Cromey, M.S. - Associate Scientific Investigator
> Dept. of Cellular& Molecular Medicine, University of Arizona
> 1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA
>
> office: AHSC 4212 email:
[hidden email]
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>
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>
>
--
George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054