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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Preparation of Slides and Tissue
- Use either Silane-prep slides or polyethylene napthalate (PEN) membrane glass slides.
- For RNA isolation, dip slides in RNase decontamination solution, wash 3 times with RNase-free water then dehydrate with a graded series of RNase-free ethanol and vacuum dry for 30 min.
- Cut tissue sections at 10 µm thickness using a cryostat and mount on the prepared RNase free slides. Keep the sections frozen during sectioning to preserve RNA quality. NOTE: Be sure that the tissue is approximately 4 mm away from the edges of the slide as the LCM cannot remove tissue that is too close to the edges of the slide. In order to remove tissue from a PEN membrane slide, ensure that the tissue is 4 mm from the left, top and bottom and 7 mm from the right side of the slide which are the dimensions of the membrane that is not attached to the slide.
- For isolation of individual cell populations using direct immunohistochemistry, section tissue onto either the Silane-prep slides or PEN membrane slides. For isolation of regions of tissue using indirect immunohistochemistry guided techniques, mount one set of sections on a Silane-prep slide for immunohistochemistry and alternate sections on either a PEN membrane slide and/or a Silane-prep slide to be used for LCM.
2. Tissue Preparation for Laser Capture – Rapid Tyrosine Hydroxylase Immunohistochemistry for Direct Laser Capture of Immunoreactive Cells
- Outline tissue with a hydrophobic pen and allow to dry.
- Fix tissue in acetone-methanol (1:1) solution at -20 °C for 10 min. NOTE: Our experience has shown that the acetone-methanol fixation resulted in much more consistent immunohistochemistry than acetone or methanol alone.
- Rinse slide in phosphate buffered saline (PBS) with 1% Triton (RNase free).
- Cover sections with 100-200 µl PBS with 1% Triton with tyrosine hydroxylase antibody diluted 1:100 with 400 U/ml RNasin. Incubate for 5-10 min.
- Rinse briefly in PBS twice and PBS-1% Triton.
- Cover tissue with 100-200 µl of goat anti-Rabbit IgG labeled with Alexa Fluor 488 diluted 1:100 in PBS-1% Triton with 400 U/ml RNasin and 50 ng/ml DAPI. Incubate for 5 min.
- Rinse 2 times in PBS then dehydrate 30 s in a graded series of RNase free ethanol (75%-75%-95%-95%-100%-100%).
- Incubate in two washes of Xylene for 1 min then 5 min.
- Remove slides from Xylene immediately prior to use for LCM and allow to air dry.