Responses: immunolocalization

Robert L. Last rll3 at cornell.edu
Tue Oct 12 06:25:07 EST 1993


The responses to my query about immunolocalization in Arabidopsis follow. 
Thanks to everyone who offered advice.  Judging from the correspondence,
there will be many people searching for their favorite Arabidopisis
proteins during the near future.
Rob Last
Boyce Thompson Institute for Plant Research

_________
 I have used polyclonal antibodies to localize the LFY protein in situ.  The
LFY transcript is rather rare, probably about one order of magnitude less
prevalent than transcripts of other homeotic genes in the same cells. 
Thus, the protein should also be reasonably low abundant.  This technique
is very simple; however, the fixation and embedding might not be suited for
every antigen.  A more gentle embedding procedure has been described by
Laurie Smith (whom I have to credit for suggesting the proteinase step) and
Sarah Hake for Kn localization.  The paraplast embedding that we use has
the advantage of not requiring a cryostat.  I really like the Vectastain
system, which is also the system chosen by most Drosophila people.

Please contact me with any questions.

Here goes:

ANTIBODY STAINING USING VECTASTAIN ELITE KIT

This protocol works for the rabbit-anti-LEAFY antibody, straight serum
diluted 1 :3000 to 1:5000.
Tissue is fixed in FAA as for in situ hybridization, embedded in paraplast,
and sectioned.  We have not experimented a lot, thus, alternative protocols
might work even better.

1. Dewax:  2 x 10 min in xylenes, under stirring.
2. Rehydrate:  2 min each 100 %, 100 %, 95 %, 70 %, 50 %, 30 % EtOH, ddH2O,
ddH2O.
3. Block endogenous peroxidase:  30 min 0.3% H2O2 (1:100 dilution of 30%
stock) in methanol.
4. Wash:  2 x 5 min PBS.
5. Protease treatment:  10 min 20 ug/ml proteinase K (Boehringer Mannheim)
in PBS (8 mg per 400 ml).
6.  Block protease:  2 x 2 min 2 mg/ml glycine in PBS (0.8 g per 400 ml).
7. Wash:  5 min PBT.
8. Block endogenous biotin:  20 min 2% goat serum (3 drops/10 ml), 20%
avidin D (4 drops/ml) in PBT/BSA.
9. Rinse: 2 x PBT.
10. Block avidin:  15 min 20% biotin (4 drops/ml) in PBT/BSA.
11. Wash:  2 x 5 min PBT.
12. Incubate with primary antibody, in PBT/BSA, either o/n at 4oC or 1 hr
at 37oC.
13. Wash:  3 x 5 min PBT.
14. Incubate with secondary antibody:  1:200 dilution (1 drop/10 ml), 2%
goat serum (3 drops/10 ml) in PBT/BSA, 30 min.
Prepare ABC at least 30 min before step 16:  20 ul A plus 20 ul B/ml PBT (4
drops plus 4 drops/10 ml).
15. Wash:  3 x 5 min PBT.
16. Incubate with ABC for 30 min.
17. Wash:  3 x 5 min PBT.
18. Prepare substrate:  50 ul DAB, 30 ul 1:100 diluted H2O2, 20 ul 2%
NiCl2/ml PBT. Monitor staining under dissecting scope.
DAB IS CARCINOGENIC.  INACTIVATE DAB CONTAINING SOLUTIONS WITH BLEACH.
19. Wash 3 x PBT.
20. Dehydrate through ethanol series, xylenes, mount in Permount.

Notes:
All steps are performed at room temperature, if not otherwise indicated.
Steps 8, 10, 12, 14, 16, 18 are done on slides and, except for 18, in a
humidified chamber (200 to 500 ul solution/slide).
DAB hydrochloride stock: 10 mg/ml in 50 mM Tris, pH 7.5, frozen at -70oC.
Vectastain Elite ABC Rabbit Kit (#PK-6101), Blocking Kit (#SP-2001) from
VectorLabs, phone # (415) 697-3600.
PBT: 0.1% Tween20 in PBS.
PBT/BSA:  1% BSA (Sigma A-4378, crystallized and lyophilized) in PBT.


+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

Detlef Weigel                           Phone:    (619) 534-7298
Department of Biology 0116              FAX:      (619) 534-7108
University of California                Internet: detlef at jeeves.ucsd.edu       
San Diego
La Jolla, CA 92093-0116

new address effective November 1:

Detlef Weigel
Plant Biology Laboratory                    Phone:    (619) 453-4100
The Salk Institute for Biological Studies   FAX:      (619) 558-6379   
10010 North Torrey Pines Road               Internet: detlef at salk-sc2.sdsc.edu
La Jolla, CA 92037
_____

I have been using a technique for
immunocytochemistry on arabidopsis based on embedding tissue in a
particular kind of methacrylate resin. I have been following cytoskeletal
antigens, such as microtubules, but other proteins have also been
successfully localized with the method. I have described this fully in 1992
Planta: 187:405 - 413. This method is particularly nice on root tips, but
large vacuolated cells, ie mature root cortical cells are not quite as
lovely. I have not had much experience with shoots. I have heard that
several labs have been using this method with good results on several other
kinds of tissue, species. In your searching around for good methods, I
don't really see any reason to prefer ones tried and tested on Arabidopsis.
Although each tissue will have its quirks, most of these methods are robust
enough to work broadly.
        If you are interested in trying the methacrylate method cited
above, please contact me if you have any other questions. There are one or
two fine points that may need a bit further explanation.
        Good luck, have fun
        Tobias Baskin
********************************                      ***************
Tobias I. Baskin                            /~~~\ 
Biol. Sci's * Univ. of Missouri            c|o o\
Columbia, MO  65211 USA                     \ = /
Tel:314-882-0173                             """
FAX 314 - 882 - 0123
baskin at biosci.mbp.missouri.edu

___________

In answer to your query about immunocytochemistry with Arabidopsis, 
I have used techniques developed in our lab for probing arabidopsis
leaves embedded and sectioned in PEG 1500 (see Robertson et al 1993, 
Plant Cell and Environment, 16, 809-818). It works fine using
antibodies against thylakoid proteins and lighting up the chloroplasts.
With a bit of luck you can visualise thylakoids inside chloroplasts.
You might need to play around with the section thickness a bit depending
on the tissue type, we normally use 7um but can go down to 3um thickness.

all the best
kevin pyke, york, uk

________


I'm sure you know this already, but our experience from yeast
immunolocalization indicates that affinity purification is well worth the
effort.  

Best of luck,

Daphne Preuss



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