Immunofl uorescence on frozen sections
PALM User Protocols
Microdissection from Carl Zeiss
ZEISS Labs
A unique service of Carl Zeiss
PALM User Protocols
2
Immunofl uorescence
Tongue epithelium
(Rat, Anti-Cytokeratin-FITC, DAPI)
Some helpful tips before starting:
To prepare sections for non-contact laser capture microdis-
section (LCM) we generally recommend MembraneSlides.
If weak fl uorescence or small objects must be detected the
MembraneSlide 1.0 PET (Order No. 415190-9051-000) is
recommendable.
(PEN membrane on Slides can lead to more
background in some fl uorescent fi lters due to its structure
and stronger autofl uorescent effects)
Note: To allow subsequent cutting and lifting a coverslip and standard mounting medium
must not be applied! Freezing media like OCT or similar may be used for sectioning
but should be kept to a minimum and have to be removed before laser cutting.
For collecting microdissected samples we recommend the
special AdhesiveCaps:
AdhesiveCap 500 opaque (Order No. 415190-9201-000) or
AdhesiveCap 500 clear (Order No. 415190-9211-000).
PALM User Protocols
Immunofl uorescence
3
Microtome cutting and antibody staining
• Tissue (e.g., frozen rat tongue) is cut as usual (8-10 μm).
• The frozen section is transferred from the blade to the warmer slide by cautious touching.
• Dry in the cryostat for 1 minute at about -20°C.
Note: Few minutes of longer drying or previous poly-L-lysine coating of the slide may
improve the adhesion of sections during the following staining steps.
• Subsequently fi x and dehydrate the section in ice-cold pure acetone for 30 seconds.
• Finally air-dry at room temperature for 2-3 minutes. Keep refrigerated till further use.
• To reduce the necessary incubation volumes draw a hydrophobic line around the sections
with a special pen (e.g., Dako Pen #S2002).
• Rehydration and blocking of unspecifi c binding is done by covering the section with a
drop of ready-to-use Protein Block Serum-Free (DakoCytomation #X0909) for 15 minutes
at room temperature.
• Pour off protein block and dip once into PBS for short washing.
• Remove excess liquid from the slide by tapping on an absorbant surface.
• Incubate with Ab/DAPI-solution for 1 hour at room temperature in a dark wet chamber.
• Pour off Ab/DAPI-solution and dip once into PBS for short washing.
• Remove excess liquid from the slide by tapping on an absorbant surface.
• Sections can now be viewed on the microscope and used for LCM. To stabilize the fl uorescent
signals a drop of PBS or VECTASHIELD may be added as cover but microdissection will only
be possible without too much liquid.
Note: FITC-fl uorescence is very sensible to bleaching without protection (often not more than
30 seconds) and therefore fl uorescent illumination time should be kept to a minimum.
The „Freeze Mode“-function of the PALM RoboSoftware will be very helpful in this context.
Preparation of Ab/DAPI-staining solution
Dilute labelled antibody (Ab) solution in PBS containing 0.5 μg/ml DAPI shortly before use
(e.g., mouse monoclonal PCK-26, panCytokeratin-FITC; #ab112114, abcam; e.g.,1:25 - 1:100).
The optimal dilution has to be found empirically for any tissue. Keep this solution in the dark
till use and during the incubation.
PALM User Protocols
Immunofl uorescence
4
Brochures and protocols
Live cells Chromosomes DNA
FISH Immunofl uorescence RNA
For questions, comments or protocol requests please contact:
ZEISS Labs
E-Mail: [email protected]
Hotline: +49 8990 9000 900
PALM User Protocols
Immunofl uorescence
5
Car
l
Zeiss MicroIma
g
in
g
Gm
bH
0
774
0
Jena,
G
erman
y
BioSciences I Location Munich
P
ho
n
e
: +4
9
8990
9000
800
Telefax: +49 8990 9000 820
E-Mai
l
: pa
l
m-in
f
o@zeiss.
d
e
www.z
e
i
ss
.
de
/mi
c
r
od
i
ssect
i
o
n
For scientifi c questions please contact
E-Mail: [email protected]
Hotline: +49 8990 9000 900
www.zeiss.de/labs
March 2010 PALM Protocol – Immunofluorescence on frozen scetions
