[Histonet] Human Specific Ab in FFPE mouse tissue

From:"Donin, Nick (NIH/NCI)"

Hello, 

I'm attempting to stain xenografted human cells in mouse brain and am not
having much success.  The cells are glioma U251 cells expressing GFP, and
I'd like to stain for both GFP and for all human cells.  We tried once
already with two antibodies and both sets came out completely clean, no
staining.  We know, at least, that our secondary, anti-mouse antibody does
not bind non-specifically to our mouse tissue, which is good.  Can someone
suggest a human specific antibody or antigen, and also an anti-GFP antibody
that works reliably in FFPE tissue.  My protocol is below.  Please email me
at doninn@mail.nih.gov   with any suggestions.
Thanks so much everyone, it is much appreciated.  Nick.  

 

PARAFFIN SECTIONS (fixed using PFA)

1.        2 X Xylene, 5 min

2.        2 X 100% ETOH, 5 min

3.        2 X 95% ETOH, 5 min

4.        1 X 70% ETOH, 5 min

5.        Proceed to step 1 for Frozen Sections OR go to Antigen Retrieval

 

ANTIGEN RETRIEVAL

1.        Heat Vector Antigen retrieval solution (working dilution) in
pressure cooker

2.        Immerse slides, close top, and heat until cap begins rocking

3.        Allow to rock for 1 minute

4.        Take cooker off heat source, allow to cool, remove slides (Go to
step two of Frozen Sections)

 

FROZEN SECTIONS

1.        Fix 12 min in 85% ETOH

2.        Wash PBS 3 x 5 min (or 3x10 dips)

3.        Quench 20 min. in 3% hydrogen peroxide

4.        Wash PBS 3 x 5 min

5.        Block 1 hr (or 20 min) in PBS + 10% Horse serum (10% frozen serum
in PBS)

6.        Add 1 antibody diluted in PBS + 2% NHS, 4 overnight, in a
humidified chamber (start with double concentration recommended for Western,
200 ul per slide.  

 

NEXT DAY

1.        Washin PBS 3 x 5 min

2.        Add 2 Antibody: Vector kit: 2 drops (100 ul) NHS, 2 drops (100
ul) antibody in 5 ml PBS, 1 hr (or 30 min) at RT.  (Make up ABC during this
incubation - Vector Kit: 2 drops A + 2 drops B in 5 ml PBS... must sit at
least 30 min at RT)

3.        Wash PBS 3 x 5 min

4.        Add ABC, 1 hr (or 30 min) at RT

5.        Wash PBS 3 x 5 min

6.        Add DAB: Vector Kit: 2 drops buffer, 4 drops DAB, 2 drops H202 in
5 ml DDH20).  Leave 30 sec. to 5 min depending on color development (monitor
visually for color change).  

7.        Stop in tap water

8.        Counterstain in Hemotoxylin (Mayers - 10 min)

9.        Wash with running tap water 2-5 min (longer washing makes stain
more blue)

10.       Dip in 95% ETOH

11.       2 x 100% ETOH 2 min

12.       2 x Xylene 2 min

13.       Permount

 

 

Nick Donin

CRTA

Neuro-Oncology Branch

National Cancer Institute

National Institutes of Health

9000 Rockville Pike

Building 35, Room 2B-203

Bethesda, MD 20892

 

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