[Histonet] sudan black information "the cliff notes"

[Carmen Contreras-Sesvold]

{Here are the "histonet" notes I saved regarding the use of sudan
black B, like I said, I have a lot to be thankful for}


By John Kiernan:

Entering autofluorescence as a search term at
http://www.histosearch.com brought up 100 hits all dated in the last 3
years. This is a problem many people have,
and the number and variety of remedies indicate that there's no
perfect solution. In one recent comparative study,
Baschong, Suetterlin and Laeng (J. Histochem. Cytochem. 49:1565-1571, 2001)
found that staining for 30 min with 0.1% sudan black B  in 70%
alcohol, after immunofluorescent staining, solved most
autofluorescence problems.
After staining the dye was washed off with a jet of a modified Hanks's
solution and then immersed in the same solution for 10 minutes.
The authors said this washing was necessary to avoid formation of a
black precipitate.



By Gayle Callis:

(In response to autofluorescent in red and green channels)
Sounds like you might be seeing lipofuschin granules, common
autofluorescent intracellular structures in brain.
To reduce autofluroescence see

Baschong W, Suetterlin R, Laeng RH.
Control of autofluorescence of archival formaldehyde-fixed,
paraffin-embedded tissue in confocal laser scanning microscopy (CLSM).
J Histochem Cytochem. 2001 Dec;49(12):1565-72.
{[PMID: 11724904}

 They describe studies using ammonia-ethanol, borohydride, and sudan
Black B alone or in combination,
with a variety of fixation and  section prep methods compatible with
immunolabeling.
You can find other  literature regarding the application of these
reagents to autofluorescence  problems.
Expect some trial-and-error to figure out what works best for your
particular specimens.

 {I have read it and found it helpful}



By Paul M.:

Autofluorescence in CNS tissue, as well as some other tissues, is
often due to the presence of lipofuscin.
Staining with Sudan Black B after immunolabeling is often effective in
suppressing such background autofluorescence.



Make up 0.l% solution in 70% ethanol.  Heat to boiling, then cool and filter.
Optimum staining time may vary somewhat from one tissue to another and
for sections of different thickness,
but 10 minutes usually provides an acceptable level of suppression,
and often complete suppression of autofluorescence.
Quickly rinse off the stain with water or buffer, several rapid
changes, and coverslip as usual.



By me:

I have stored the above solution for a month now at RT.
And have used it on 10-20 micron thick sections and have optimal reduction
of green autofluorescence in about 5 minutes of exposure. I rinse my slides
in a Copeland jar filled with PBS on a platform rocker for 3 minutes and
coverslip with prolong as usual.