Immunohistochemistry of Mouse Brain Tissue Sections for Targeting Dopamine Neurons

Samenvatting

Source: Kummari, E., et. al., Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area., J. Vis. Exp. (2015)

In this video, we demonstrate the immunohistochemistry protocol for dopamine neurons in brain tissue sections for subsequent laser capture microdissection.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Preparation of Slides and Tissue

1. Use either Silane-prep slides or polyethylene napthalate (PEN) membrane glass slides.
2. 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.
3. 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.
4. 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

1. Outline tissue with a hydrophobic pen and allow to dry.
2. 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.
3. Rinse slide in phosphate buffered saline (PBS) with 1% Triton (RNase free).
4. 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.
5. Rinse briefly in PBS twice and PBS-1% Triton.
6. 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.
7. Rinse 2 times in PBS then dehydrate 30 s in a graded series of RNase free ethanol (75%-75%-95%-95%-100%-100%).
8. Incubate in two washes of Xylene for 1 min then 5 min.
9. Remove slides from Xylene immediately prior to use for LCM and allow to air dry.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Silane prep-slides	Sigma-Aldrich, St. Louis, MO	S4651
95% Ethanol-RNase Free	Sigma-Aldrich, St. Louis, MO	E7148
100% Ethanol-RNase Free	Sigma-Aldrich, St. Louis, MO	E7023
Rnase Free Triton	Sigma-Aldrich, St. Louis, MO	T8787
Anti-Tyrosine hydroxyase antibody	EDM Millipore, Billerica, MA	Ab152
Alexa Fluor 488 goat anti-rabbit IgG	Life Technologies, Grand Island, NY	A-11008
RNaseZap	Life Technologies, Grand Island, NY	AM9780