Tissue Recombinants to Study Extracellular Matrix Targeting to Basement Membranes - Methods in Molecular Biology: Extracellular Matrix Protocols - page 297

2. Dissolve 1 g agar (Bacto-Agar, Difco Laboratories, E. Molesley, Surrey, UK) per

100 mL Hank's solution in a boiling water bath; sterilize solution by autoclaving.
Once cooled, the solution can be kept at 4 ° C until use.

3. The day before the experiment, prepare the number of dishes required containing

the gelified medium. Melt the agar solution in a boiling water bath. Then rapidly
add to the Ham's F10 solution (+ antibiotic) the adequate amount of melted agar.
Mix thoroughly.

4. Dispense approximately 2 mL Ham's F10/agar/antibiotic mixture into 3-cm

diameter culture dishes.

5. Allow to gel overnight at 4 ° C.

2.2. Pregrafting Culture: Preparation
of Enriched Gelified Medium
1. Prepare chick embryo extracts by mechanical homogenization of 9-d-old

embryos. Clarify by centrifugation. Store by aliquots at -20 ° C.

2. Mix 9 mL of Dulbecco's modified Eagle's medium (DMEM; Gibco, Life Tech-

nologies), 1 mL of 9-d-chick embryo extract, 2 mg/mL gentamicin to 2.5 mL of
agar solution (as prepared in Subheading 2.1.2. ). To be prepared the day before
the experiment. Keep at 4 ° C.

2.3. Dissociation of Embryonic Intestines
1. Collagenase solution make fresh each time : dissolve 2.5 mg collagenase A
(0.5 U/mg) from Clostridium histolyticum (Boehringer Mannheim, Germany) in
10 mL CMRL 1066 medium (Gibco, Life Technologies) ( see Note 1 ).

2. Blocking solution: mix Ham's F10 medium to newborn bovine serum (1:1)

(Gibco, Life Technologies).

2.4. Immunocytochemistry
1. 0.1 M phosphate buffered saline solution (PBS): dissolve 13.6 g KH 2 PO 4 and

14.2 g Na 2 HPO 4 , in 1 L H 2 O. Adjust pH to 7.2. Store at 4 ° C.

2. Antibody dilution solution: add 0.01% NaN 3 as a preservative agent to the 0.1 M

PBS solution.

3. Mounting and antifading solution:

• solution A: dissolve 0.136 g KH 2 PO 4 and 0.876 g NaCl in 100 mL water.

Adjust pH to 7.4. Store at 4 ° C.

• solution B: dissolve 1.59 g Na 2 CO 3 and 2.93 g NaHCO 3 in 100 mL water.

Adjust pH to 9.0. Store at 4 ° C.

• mix 10 mL of solution A with 100 mg p-phenylenediamine (Sigma, Madison,

WI): caution -toxic and carcinogenic. Adjust pH to 8.0 with solution B. Then
add 90 mL glycerol. Store in aliquots at -20 ° C.

• the solution has to be thrown away when it turns violet.

Note: All solutions and materials must be sterile; manipulations must be
done under sterile conditions.