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Using WETSEM Capsules for Biology

Purpose:
!
Using the WETSEM capsules is required for biological samples and allows for
the imaging of samples that are in solution or wet. All Imaging is done through Dr. Fodor
and his laboratory assistants.

Materials:
!
QX-102 capsule, QX imaging buffer, double distilled water, QX aspirator tips,
MP-10 multi-well plate, fibronectin/gelatin/Poly-L-lysine capsule coating, staining agent
(optional).

Quick Tips:
The capsules are fully sealed when the wings of the liquid dish and sealing stub are
aligned
Take extreme care with the capsule membrane. Do not touch, set facing down unless
on multi-well plate, and use caution when filling even when using QX aspirator tips.
Unused capsules have a shelf life of 18 months from production date and should be
stored in a dry and dark environment at room temperature.

Preparing Samples:
NOTE: QX-102 imaging buffer is formulated to reduce damage to the sample and is
recommended for use with any imaging done with the QX-102 capsule for best result.
a. If live biological sample images are wanted, sample growth can be done in the
QX-102 capsules or grown in normal laboratory conditions and then transplanted
onto the capsules.
b. Tissues and muscle can also be placed into the QX-102 for imaging.
c. Samples can also be fixed and stained (optional) for imaging.
d. Removing liquids does not require special tool, just extreme caution to not touch
the membrane with pipet tip, just as with filling liquid dish. (As shown above)
NOTE: Staining is an optional step and is not required to image biological samples,
however, using one of the stains below is recommended, but any stain with heavier
elements like phosphorus will help with image quality and structure clarity.

Membrane Coating:
I.

Fibronectin Coating

II.

- Can be substituted with collagen, laminin or a mixture of ECM molecules


1.

Dilute Fibronectin with PBS to a final concentration of 0.01%

2.

Apply 15 l to the liquid dish and incubate for 30 minutes at room temperature

3.

Remove the solution and wash twice with PBS

4.

Wash twice with growth media

5.

Keep the liquid dish filled with medium until seeding cells

6.

Plate cells on fibronectin within one day from coating

Gelatin Coating
1.

Dilute to a recommended starting concentration of 0.1% w/v with using a


higher concentration to improve attachment for some specimens

2.

Filtration sterilization or UV sterilization when coated is recommended

3.

Apply 15 l to the liquid dish and incubate for 30 minutes at room temperature

4.

Remove the solution and wash twice with PBS

5.

Keep the liquid dish filled with PBS until seeding cells

6.

Plate cells on gelatin within one day from coating

III. Poly-L-lysing Coating


- Poly-L-lysine is recommended for the attachment of non-adherent cells or
microorganism. It is not the first choice for other cells since it interferes with the
growth and other functions of cells.
1.

Apply 15 l of 0.1% Poly-L-lysine solution to the liquid dish and incubate for
one hour at room temperature. Longer incubation times, up to overnight, also
give good results.

2.

Remove the solution and rinse the liquid dish twice with distilled water.

3.

Water can be removed and the dry liquid dishes for later use, or kept wet.

Fixation:
- Three example protocols for fixation, however, your normal laboratory fixation will work
I.

II.

Glutaraldehyde Fixation
1.

Dilute Glutaraldehyde to a 2% final concentration in PBS

2.

Wash the sample four times with PBS

3.

Incubate with 2% solution at room temperature for 30 minutes

4.

Wash four times with PBS

Paraformaldehyde Fixation
1.

Dilute Paraformaldehyde to a 4% final concentration in PBS

2.

Wash the sample four times with PBS

3.

Incubate with 4% solution at room temperature for 15 minutes

4.

Wash four times with PBS

III. Methanol Fixation


1.

Wash sample four times with PBS

2.

Change to pre-cooled (-20C) 100% Methanol

3.

Incubate for five minutes at 4C or room temperature

4.

Wash four times with PBS

Staining Protocols:
There are many staining agents that can be used in the SEM, some of which can be
hazardous. Such stains as Uranyl Acetate and Osmium Tetroxide can produce very
clear images but have significant hazardous material handling protocols. They will be
outlined, however, the use and disposal steps may be different or require special
training. The department of environmental health and safety must be contacted for
proper use, training and ordering of these stains. Although they are the standard of use
for many imaging studies they are in no means the only stains that can be used with the
SEM.

I.

Osmium Tetroxide Staining

WARNING: Osmium Tetroxide is volatile and toxic. Work should all be performed in a
fume hood using gloves and protective clothing. Handling and waste disposal should
be done according to the guidelines of the local authorities and environmental health
officer
- Osmium Tetroxide is an excellent stain for lipide in membranous structures and
vesicles. Some intercellular structures can be visualized also.

II.

1.

Wash sample four times with PBS

2.

Fix cells for 30 minutes

3.

Wash four times with PBS

4.

Wash four times with distilled water

5.

Prepare a 0.1% Osmium Tetroxide solution

6.

Incubate the sample for 30 minutes with 0.1% solution

7.

Wash four times with distilled water

8.

Ready to image

Phosphotungstic Acid Staining (PTA)

- PTA positively stains charged structures such as basic proteins associated with
nuclear DNA and nucleoli. PTA also intensely stains mitochondrial matrix.
1.

Wash the sample four times with PBS

2.

Fix the cells for 30 minutes

3.

Wash the sample three times with PBS

4.

Wash three times with double distilled water

5.

Incubate the sample with 2% PTA for 30 minutes

6.

Wash five times with double distilled water

7.

Ready to image