Free-floating Mouse Brain Immunohistochemistry

Remove PFA-fixed tissue from storage solution and add to mesh bottom netwell insert inside of 12 well plate that is filled with 0.22 μm filtered 1x PBS.

Place the 12 well plate with the tissue on a horizontal shaker and wash tissue 3x 5 min at approx. 150 rpm, moving the netwell insert to the next well down after each 5 min period to immerse the tissue in new 1x PBS. Room temperature

Per well of tissue make a minimum of 3.5 mL of blocking solution, consider this is needed for blocking, primary and secondary antibody incubation. All reagents should be 0.22 μm filtered:

1x PBS

5% Normal Goat Serum

2.5% Bovine Serum Albumin

0.2% Triton-X

Make in excess and keep on ice. Keep at 4 degrees celcius overnight. 4°C

In a new 12 well plate, add 2-3 mL per well of blocking solution and transfer the washed tissue sections inside their netwell inserts into these wells. Incubate for 1h - 2.5 h on horizontal shaker at approx 150 rpm. Room temperature

Meanwhile, dilute primary antibodies in blocking solution to appropriate concentrations and keep On ice .

E.g.

• Mouse monoclonal anti pS129 (81A) (ab184674) @ 1:750

Add approx 250 ul of primary antibody solution to the approriate number of wells of a 24 well plate for the number of brain sections.

When blocking is finished, move brain tissue sections from the netwell inserts to their appropriate primary antibody well using a fine paintbrush, being careful not to destroy the tisssue.

Incubate on horizontal shaker at approx. 150 rpm overnight. 4°C

Prepare a new 12 well plate with clean netwell inserts and fill all wells with 0.22 μm filtered 1x PBS.

Transfer brain tissue sections from 24 well plate into netwell inserts inside 12 well plate and wash 4x 10 minutes on a horizontal shaker at approx. 150 rpm. Room temperature

Meanwhile, make appropriate dilutions of secondary antibody in blocking solution. Keep away from light and keep On ice .

E.g.

• Donkey anti-Mouse Alexa Fluor 568 (A10037) @ 1:500

Add approx 250 ul of secondary antibody solution to the approriate number of wells of a 24 well plate for the number of brain sections.

When washing step is finished, move brain tissue sections from the netwell inserts to their appropriate secondary antibody well using a fine paintbrush, being careful not to destroy the tisssue.

Keep the well plates covered from now to avoid bleaching of fluorophores.

Incubate 24 well plate on a horizontal shaker at room temperature at approx. 150 rpm for 1 h - 2.5 h. Room temperature

Transfer brain tissue sections from 24 well plate into netwell inserts inside 12 well plate and wash 4x 10 minutes on a horizontal shaker at room temperature at approx. 150 rpm. Room temperature

Plasma-clean microscope cover slips in Argon plasma for 15 minutes.

Using a fine paintbrush, transfer brain tissue sections from 1x PBS onto a microscope slide, using excess 1x PBS to mount multiple sections next to each other and making sure they are not folded over.

Leave sections on microscope slides to dry out in the dark (they will turn white).

Add approx. 150 uL of mounting media on top of each tissue section and apply the plasma-cleaned cover slip on top of the tissue, closing the slide.

Leave the mounting media to dry in the dark.

Seal the edges of the coverslip with nail varnish and let dry for approx. 30 minutes in the dark.

Immediately take finished microscope slides to imaging, avoiding unecessary light exposure.

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