[Histonet] RE: goat anti Rabbit Alexa 594 woes.

[gayle callis]

You wrote: 

 

I would like to get your keen ideas about two issues I am facing now in my
immunohistochem. procedure:

 

1- I am using goat anti rabbit Alexa 594 (red) as secondary antibody for
detecting C-Fos in rat nervous system. When I am looking at the slides, it
seems always I have some residues of my

Alexa remained on the surface all over the section. I've added one extra
quick wash in every washing steps (Using PBS+TX100). But still they are
exist. Is anybody has suggestion to improve the quality of the picture and
get ride of these residues?

 

 

Leila Etemadi

M.Sc., Ph.D Candidate

Neuronano Research Center (NRC)

Lund University, BMC F10

Sweden

 

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This is only a reply to #1 inquiry about "residues" on tissue after staining
with antibody-fluorophore conjugate.   What you have is deposition of
fluorophore-protein aggregates from antibody that tends to break down over
time and this is unavoidable and quite normal.  

 

Solution is dilute your goat antiRabbit Alexa 594 to working concentration
in micro centrifuge tubes, and spin down diluted antibody in a desk top
micro centrifuge a couple of minutes or so just before applying to the
tissue section.   Pipette the spun antibody carefully, keeping pipette tip
out of bottom of micro centrifuge tube,  to avoid remixing the fluorescent
aggregates and apply to section.   Every time I did not spin my diluted
antibody to take a shortcut in time (the lazy approach),  I ended up with
what we called "glowing garbage".     More rinses or longer rinsing did not
solve this problem as the aggregates tend to sit on the sections and slide.


 

Over the years of working with fluorophore conjugated antibodies, there some
things I did faithfully to keep sections free of "glowing garbage" 

 

1. Rinse buffer contained detergent, with our preference 0.025% Tween 20 and
NO protein carriers e.g. no BSA or normal serums.   Triton X 100 is ok to
use.   This rinse buffer was used to make up antibody diluent and blocking
reagent.   

  

2.  The final rinse buffer had no additives, just pure buffer before I cover
slipped with Prolong Gold Antifade reagent from Molecular Probes. I did more
final rinses X5 to make sure all antibody-fluorophore is removed. 

 

Take care

 

Gayle M. Callis

HTL/HT/MT(ASCP)