3.2.3 Embedding
of Matrigel Plugs in Paraffin
- Transfer the fixed plugs into labelled tissue cassettes. Labelling
has to be performed with a lead pencil (seeNote 53). Collect
the cassettes in a basket (belonging to the tissue processing
equipment), and put it into a container filled with tap water.
Close the basket with the metal lid, and place the container
under gently running tap water for 2 h. - Take out the basket from the container, let the water drip off,
and wipe off the remaining water with a dry tissue (seeNote
54 ). - Insert the basket into a tissue processing equipment, and per-
form dehydration of samples according to the following proto-
col: 21 h in 70% ethanol, 1 h in 96% ethanol, 22hin96%
ethanol, 22 h in 99% ethanol, 1 h in ethanol:xylene 2:1, 1 h
in ethanol:xylene 1:2, 1 h in xylene, 2 h in melted paraffin, and
another 3 h in a second vessel with melted paraffin. - Transfer the tissue cassettes to an embedding station. Place the
Matrigel plugs with the help of a warm forceps into a metal
casting mold with the flat side to the bottom. Fill the mold with
melted paraffin and let the plug sink for 5 s (seeNote 55). Put
the labelled part of the tissue cassette on top of it, add more
paraffin, and transfer the whole mold to the cold plate to
solidify the paraffin. After 30 min, remove the paraffin block
from the mold, and store it at room temperature. - Obtain sections of the Matrigel plugs for staining using a
microtome. Cut the paraffin block down till the plug is visible
(seeNote 56). Discard 500μm and start sectioning with four
5 μm sections (level 1). Repeat this step for up to 12 more levels
with 100μm distance in between. Transfer sections into a water
bath at room temperature using a small paintbrush, and keep
them there until mounting (seeNote 57). - For mounting, transfer sections into a 37C water bath using a
glass slide (see Note 58). Let them float on a labelled
polylysine-coated glass slide, and fix them with the help of a
needle. Mount two sections on one slide. Dry sections at room
temperature for 24 h. They can be stored at room temperature
or at 2–8C in slide storage boxes for several years.
3.2.4 Analysis
of Angiogenesis in Matrigel
Plugs by CD31 Staining
- Rehydrate the Matrigel plug sections by incubating them 3
10 min in xylene, followed by subsequent 5 min incubations in
99%, 96%, 70%, and 50% ethanol and ddH 2 O. - For antigen retrieval, transfer the slides into citrate buffer
(prewarmed for 5 min in a microwave, 630 W), and boil
them for 12 min in a microwave set to 630 W. Afterwards,
leave the slides in the buffer until they cool down to room
temperature.
530 Katrin Spengler et al.