double fluorescent staining

From:Carol Bobrowitz

My tissue is FFPE rat cremaster.

Stained with SMA with Texas red detection and FITC labeled Griffonia
(Bandeiraea) Simplicifolia BS-1.

The fluorescent staining is coming from RBC's and with both the GS1 and the
SMA,
the main staining is in the lumens, not the walls,
there is some SMA positive on the largest vessels.

When looking at the slides in detail, the green and red fluorescence always
match, and that should NOT be the case and the RBC's should not be the
strongest fluorescence.

The positives and the negatives are the same.  (the negatives only had
secondary Texas red applied)

My guess the RBC's are picking up staining because the titer used for the
texas red and FITC labeled primary are too strong and need to be taken out
further.

I do not have access to these fluorescent stained slides and am going by
just what I've been told.

I have never done brown IHC staining with GS1.  Does this lectin stain the
same area's that SMA does?
I'm using my memory of Ulex as a guide.

I'm using SMA from DAKO, monoclonal mouse anti Human Smooth Muscle Actin,
clone1A4.  The specification sheet says the antibody cross-reacts with the
alpha smooth muscle actin-equivalent protein in chicken cow and rat.

I have used this SMA on rat kidneys and they were very clean with good brown
staining.
I never used heat-induced epitope retrieval with this antibody.  I now
notice the spec sheet recommends this.

Should I ask the researcher to supply a SMA that is anti-rat?
Using GS1 unconjugated antibody with a FITC secondary achieve better
staining?

Any help from Histo Land would greatly be appreciated.

Thank you,
Carol Ann Bobrowitz
Physiology & BRI Histology Core Laboratory
Department of Physiology
Medical College of Wisconsin
Milwaukee, Wisconsin
(414) 456-8179
FAX (414) 456-6546
cbobrowi@mcw.edu









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