Palm microbeam laser micro dissector

Immunohistochemical stainings were performed on formalin-fixed, paraffin-embedded (FFPE) and frozen sections of various human tissues. Antigens were retrieved by heating the sections in 0.01 M citrate buffer (pH 6.0) 4 x 5 min. (at 700 W) in a microwave oven. Non-specific tissue and endogenous peroxidase reactivities were blocked with 10% BSA and 3% H₂O₂, respectively. Sections were incubated at 4°C overnight with the rabbit primary polyclonal anti-CRNDEP antibody, specific to the Epitope 2 (1:800, Abgent, Inc.). Biotinylated secondary goat anti-rabbit antibody (1:1500), peroxidase-conjugated streptavidin (1:500) (both from Immunotech Laboratories, Inc., Monrovia, CA, USA) and DAB were used as a detection system. Specificity of the immunohistochemical reaction was determined by using the synthetic Epitope 2 (originally utilized for rabbits' immunization during the antibody development) as a blocking peptide. The anti-CRNDEP antibody and the blocking peptide (1:3) were incubated for 2h before application. The sections were counterstained with hematoxylin and then examined under the Olympus BX41 light microscope equipped with the DP70 camera (Olympus, Shinjuku, Tokyo, Japan) or the PALM MicroBeam Laser Microdissector (Carl Zeiss AG, Oberkochen, Germany).

Paraffin-embedded adult F. hepatica (prepared as described by Hanna et al. [55 (link)]) were cut into thick (10 µm) sections (to maximise tissue recovery), placed on polyester membrane steel-framed slides (Leica Microsystems Ltd., Newcastle Upon Tyne, UK), and lightly stained in 1% (w/v) toluidine blue for 2 s at room temperature (18–21 °C) to allow visualisation of the tissue. For LMD, the gastrodermal cells and tegumental syncytium were excised from the tissue sections using an ultraviolet laser on either a LMD7 (Leica Microsystems Ltd., Newcastle Upon Tyne, UK) or PALM MicroBeam Laser Micro-dissector (Zeiss) system and collected in 0.5 mL microcentrifuge tubes. LMD consisted of four independent experiments.