5/30/97

Why is it that when I do double immunostaining with two

successive monoclonals I get inconsistant results with 'stains'

that are

fine by themselves? If I do a polyclonal followed by a

monoclonal, no

problems. What is the story? Are there ways around this?

Thanks in advance!

Geoff

--

***************************************************************

Geoff McAuliffe, Ph.D.

Neuroscience and Cell Biology

Robert Wood Johnson Medical School

675 Hoes Lane Piscataway, NJ 08854

voice: (908)-235-4583; fax -4029 e-mail: mcauliff@umdnj.edu

***************************************************************

I have been discussing an immunolabeling problem with Geoff

McAuliffe. The

original post was on the Microscopy Listserver a few days ago.

The basic

problem is one of double immunolabeling with mouse monoclonals.

The following message gives additional details on the labeling

procedure.

Since the original post, I have seen one response that suggested

introducing

a saturating step with fab fragments after the first labeling

run.

If you have any suggestions or thoughts on other things to try,

please

correspond directly with Geoff at the address in the header.

Many thanks, and greetings from sunny (and HOT!) Arizona.

Bob Chiovetti

(RCHIOVETTI@aol.com)

---------------------

Forwarded message:

From: mcauliff@UMDNJ.EDU (Geoff McAuliffe)

To: RCHIOVETTI@aol.com

Date: 97-06-02 14:04:47 EDT

Thanks for responding to my querry. I should have been more

specific re the objects of my study.

Mouse CNS, light microscopy, paraformaldehyde fixation, 20

micron

frozen sections, stained free-floating with mouse monoclonal anti

Mac-1

(macrophages and microglia) with a Vector ABC Elite kit

(biotinylated

secondary, etc.). Mount sections on slides stain with mouse

monoclonal

anti-bromodeoxyuridine after trypsin unmasking (hence mounting on

slides)

also with a Vector ABC Elite kit. Done individualy the results

are fine

but when combined the staining with the second AB is greatly

reduced and

very spotty.

The trypsin is essential after formalin fixation, and Mac1

does

not survive any fixation that allows omission of trypsin

(alcohol). I

don't think I can do the Bromodeoxyridine first since the trypsin

will

digest the cell surface marker Mac-1 detects. Hot buffer antigen

retrieval does not give good results with bromodeoxyuridine.

If I use a rabbit polyclonal for GFAP at the first antibody

I get

beautiful double staining but of two seperate populations of

cells so I

am not sure if my problem is trying to use two mouse monclonals

to stain

two things in one cell or two things on one section. I am getting

the

idea that the secondaries are the problem? so perhaps one or both

primaries linked to gold? I do need to see two colors, though.

Thanks in advance!

I think it should work fine if both monoclonals are directly

conjugated.

But if you are using conjugated secondaries you will get your

second

primary sticking to your first secondary. You will also get your

second

secondary sticking to your first primary. So you will get various

states

of crossover. I think after you put on your first primary and

secondary,

block with 10x excess of anti-mouse Fab fragments to bind up all

the

free sites should work.

Bob Underwood

Morphology Core

U of W

Seattle, WA, USA