- Inclusion of positive (for instance, bFGF) and negative con-
trols (no treatment, inhibitors only) in an experimental series is
essential. - Heparin supports efficient binding of VEGF 165 to the VEGF
receptors. - Be careful to fill the syringe slowly and to avoid bubbles.
Matrigel is highly viscid. - Keeping Matrigel at room temperature 5 min before injection
leads to a faster polymerization in vivo and helps to prevent
leakage. - The plugs can cause an inflammatory response, which may
interfere with the effect of the added compounds on angiogen-
esis. Occasionally, the Matrigel plugs can be absorbed. - We obtain best whole plug CD31 stainings when plugs are
processed with zinc fixative compared to 4% PFA. - Ball pen or marker labels will disappear when the cassette is
treated with solvents at a later stage. - Remaining water will dilute the ethanol, in which the basket
will be transferred afterwards. - Use a warm forceps if the plug has moved and needs to be
repositioned. If the plug is placed on the bottom of the mold, it
will be located on top of the paraffin block, which is of advan-
tage for cutting sections. - The Matrigel plug can be macroscopically distinguished from
the normal tissue. - At room temperature the section will not stick to the slide.
- Fragmentation of the sections may be due to too high temper-
ature of the water bath. - We use a volume of 150μL of blocking solution or diluted
antibody per slide and 1 mL of PBS per washing step when
using the Shandon Sequenza® immunostaining center
together with the Shandon coverplate technology. - Pressing carefully but firmly on the coverslip during embed-
ding gives a flatter surface which helps with focusing on the
microscope. - Slides with paraffin-embedded sections can be stored at 2–8C
for several years in slide storage boxes. Long-term storage may
reduce the staining intensity for certain antigens. - The stitching settings should be optimized before starting
imaging.
536 Katrin Spengler et al.