Ldm 6000

A Leica LDM 6000 was used to take images of tissue sections mounted on membrane or glass slides. The software, Leica laser microdissection V6.7.1.3952, was used to control the microscope when taking pictures of selecting areas for laser capture microdissection and cutting. A hardware modification was made to the collection unit allowing for samples to be collected into two strips of 8 PCR caps (VWR, Oslo, Norway). The Leica software was used to design a standard sampling pattern of 96 circles, each with an area of about 25,000 μm², placed on an 8 × 12 grid (see Figure 1). The same grid was sampled on all membrane sections. Observation of the in-between samples placed on glass slides, allowed the grids to be placed in the position corresponding to the previous sampling. 20 μl of a collection solution (1xThermopol buffer with Proteinase K (0.27 μg/μl) was added to each cap in the inverted strips. After cutting and collecting the selected areas by laser capture microdissection, the strips (with collection liquid and tissue) were mounted onto a 96-well PCR plate (Axygen, VWR, Oslo, Norway). The plate was briefly centrifuged and incubated at 56°C for 30 min. Deactivation of proteinase K was achieved by raising the temperature to 95°C for 1 min. One microliter of incubated solution was used as template for the first round PCR.

A Leica LDM 6000 was used to take images of tissue sections mounted on membrane or glass slides. The software, Leica laser microdissection V6.7.1.3952, was used to control the microscope when taking pictures or selecting areas for laser capture microdissection and cutting. A hardware modification was made to the collection unit allowing for samples to be collected into two strips of 8 PCR caps (VWR, Oslo, Norway).
20 μl of a collection solution (1×Thermopol buffer with Proteinase K, 0.27 μg/μl) was added to each cap in the inverted strips. After cutting and collecting the selected areas by laser capture microdissection, the strips (with collection liquid and tissue) were mounted onto a 96-well PCR plate (Axygen, VWR, Oslo, Norway). The plate was briefly centrifuged and incubated at 56 °C for 30 min. Deactivation of proteinase K was achieved by raising the temperature to 95 °C for 1 min. One microliter of incubated solution was used as template for the first round PCR (see above).