Sectioning : Cut sections into O.1M PB with 0.01% sodium azide. Store well covered sections in the refrigerator.

To ensure consistency in the reactions for each immuno run, solutions should be well mixed before adding to the wells. (Typically this means making them up ahead of time and letting them mix on the shaker table before use.)

Use about 9 mls. per well for the steps using cell wells. Use about 310 mls per well for steps using bubble wells.

Be compulsive about preventing cross-contamination with other antibodies: -Track on and clean each spill

- Work on clean surfaces with clean dishes, brushes, gloves, etc. - Prevent contamination of pipet men - Keep everything covered

All steps are on the shaker table at room temperature unless otherwise indicated.

The recipes below are for 32 brains, 6 sections per brain + 1 no-primary control section = 193 bubble wells and 33 cell wells)

Transfer sections to mesh bottomed dixy cups placed in cell wells containing 0.1 M PB (or 10 mM PBS). Sections per one brain per antibody can be placed together in one cup.

(1) Inactivate endogenous peroxidase

Transfer dixy cups with sections to cell wells containing 0.3% hydrogen peroxide in 10mM phosphate buffered saline (PBS) for 30 min.

30 ml 3% H₂0₂

270 ml PBS

(2) Rinse in 10mM PBS (aka "working PBS")-- 2 X 10 min.

(3)Transfer dixy cups to block solution*-- 1-2 hours.

2% horse serum, 0.1% bovine serum albumin, 0.2% Triton X-100 in PBS

14.4 ml 10% Triton-X

7.2 ml Horse serum

0.36g Bovine serum albumin

338.4 ml PBS

(4) While sections are in block solution, make up primary antibody solution:

300 ml 20% anti-MAP2

59.7 ml Block solution

Fill bubble wells with 310 ml antibody solution per well. Transfer sections from dixy cups into bubble wells.

Make sure sections are unfolded and completely covered in solution.

Incubate at 4^oC in a humidity chamber (e.g., sealed tuperware lined with wet lab diaper) for 4 days.

Day 2 (about 6 hours-ish total time)

Make up secondary antibody solution before beginning the rinses below.

1:200 secondary antibody (anti-mouse made in horse), 2% serum in PBS no azide:

300 ml anti-mouse made in horse

1.2 ml Horse serum

58.5 ml PBS with no sodium azide

(1) Transfer sections to dixy cups in PBS. Rinse 2 X 5 min. PBS

1 X 5 min. PBS with 2% horse serum (6 ml horse serum + 294 ml PBS)

(2) Transfer sections into bubble wells containing secondary antibody (310 ml per well) - 2 hr

Begin preparing ABC solution while sections are in 2ndary. ABC must be prepared and agitated on the shaker table for at least 45 min prior to use ABC solution:

12 drops reagent A

12 drops reagent B

60 ml PBS with no azide

(3) Transfer sections to cell wells containing PBS with no azide. Rinse 3 X 5 min. PBS

(4) Transfer sections to bubble wells containing 310 ml ABC solution.

Incubate at room temperature for 2 hrs.

(5) Transfer sections to cell wells. Rinse

1 X 5 min. PBS no azide

2 X 5 min. Tris Buffer

(6) DAB/NAS

Prepare nickle ammonium sulfate (NAS) solution:

2.08g NAS in 75ml Tris buffer

Prepare diaminobenzidine (DAB) solution:

150 mg DAB in 225 ml Tris buffer

Filter DAB

Filter NAS into DAB solution shortly before use

Add 100 ml 3% hydrogen peroxide to DAB-NAS just before use.

Let it stir on the stir plate for a few seconds (about 10) and quickly add to cell wells.

Transfer cups to DAB/NAS with H₂0₂ and visually moniter the reaction

(7) Rinse sections at least 5 X 5 min. in buffer.

(Tris or PBS with or without azide, whatever you have left)

Sections may be stored overnight or a bit longer well covered in the final rinse solution in the refrigerator.